Macrocyclic benzodiazepine dimers, conjugates thereof, preparation and uses

ABSTRACT

Macrocyclic benzodiazepine dimers having a structure represented by formula I 
     
       
         
         
             
             
         
       
     
     where A and B are independently according to formulae Ia or Ib 
     
       
         
         
             
             
         
       
     
     and the other variables in formulae I, Ia, and Ib are as defined in the application. Such dimers are useful as anti-cancer agents, especially when used as the drug component in an antibody-drug conjugate (ADC).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 62/183,350; filed Jun. 23, 2015; thedisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to macrocyclic benzodiazepine dimers,dimer-linker compounds derived therefrom, and conjugates thereof, andmethos for their preparation and use.

Some naturally occurring cytotoxins, such as tomaymycin and anthramycin,contain a benzodiazepine ring system. Reflecting the additional presenceof a pyrrolidine ring fused to the diazepine ring, these compounds areoften referred to as pyrrolobenzodiazepines, or PBDs.

PBDs possess antibiotic and antitumor activity, the latter trait leadingto interest in them as anticancer drugs. Mechanistically, PBDs bind tothe minor groove of DNA in a sequence selective manner and alkylate theDNA. The structure-activity relationship (SAR) of different substituentshas been studied (Antonow et al. 2010; Thurston et al. 1999).

Additional studies have shown that PBD dimers also show special promiseas anticancer agents. The core structure of a typical PBD dimer can berepresented by formula A-1, where X is a bridging group connecting thetwo dimer halves.

As with monomeric PBDs, the dimers are DNA minor groovebinder-alkylators. Being bifunctional, alkylation results incross-linked DNA, making DNA repair more difficult. (DNA alkylationoccurs via the imine group. PDBs having one of the imine groups reducedcan still alkylate DNA, but cannot crosslink it. They are stillbiologically active, albeit generally less so.) For a review on theevolution of PBDs as antitumor agents, from naturally occurring monomersto synthetic monomers to synthetic dimers, see Hartley 2011.

The SAR of PBD dimers has been explored via substituents on the A/A′ andC/C′ rings, unsaturation in the C/C′ rings, the structure and length ofthe bridging group X, and the oxidation or reduction of the imine doublebonds in rings B/B′, and combinations of such features. See Bose et al.1992, Gregson et al. 1999, Gregson et al. 2001a and 2001b, Gregson etal. 2004, Gregson et al. 2009, Hartley et al. 2012, Howard et al. 2007,Howard et al. 2009a. Howard et al. 2010, Howard et al. 2013a and 2013b,Liu et al. 2007, Thurston et al. 1996, Thurston et al. 2006, andThurston et al. 2008. Most PBD dimers are joined via an 8/8′ bridge asshown above, but a 7/7′ bridge also has been disclosed (Howard et al.2009b).

A type of anticancer agent that is generating strong interest is anantibody-drug conjugate (ADC, also referred to as an immunoconjugate).In an ADC, a therapeutic agent (also referred to as the drug, payload,or warhead) is covalently linked to an antibody whose antigen isexpressed by a cancer cell (tumor associated antigen). The antibody, bybinding to the antigen, delivers the ADC to the cancer site. There,cleavage of the covalent link (refered to as the linker) or degradationof the antibody leads to the release of the therapeutic agent.Conversely, while the ADC is circulating in the blood system, thetherapeutic agent is held inactive because of its covalent linkage tothe antibody. Thus, the therapeutic agent used in an ADC can be muchmore potent (i.e., cytotoxic) than ordinary chemotherapy agents becauseof its localized release. For a review on ADCs, see Schrama et al. 2006.

PBD dimers have been proposed as the drug in an ADC. Attachment of thelinker connecting to the antibody can be via a functional group locatedin a C/C′ ring, the bridging group X, or by addition across the iminegroup in a B/B′ ring. See Bouchard et al. 2013, Commercon et al. 2013aand 2013b, Flygare et al. 2013, Gauzy et al. 2012, Howard 2104a-2014e,Howard et al. 2011, Howard et al. 2013c and 2013d, Howard et al.2014a-2014d, Jeffrey et al. 2013, Jeffrey et al. 2014a and 2014b, andZhao et al. 2014.

Another type of benzodiazepine dimer also has been proposed as a drug inADCs. Structurally, this type may be viewed as a PBD dimer furtherhaving a phenyl ring fused to each of C/C′ rings, as shown in formulaeA-2 and A-3. See Chari et al. 2013, Li et al. 2013, Fishkin et al. 2014,Li et al. 2014.

Benzodiazepine compounds having other ring systems, such as atetrahydro-isoquinolino[2,1-c][1,4]benzodiazepine, also have beendisclosed. Kothakonda et al. 2004.

Full citations for the documents cited herein by first author orinventor and year are listed at the end of this specification.

BRIEF SUMMARY OF THE INVENTION

This invention provides novel benzodiazepine dimers, having both an 8/8′bridge and a 7/7′ bridge, to form a macrocyclic ring structure, asrepresented by formula I:

wherein

-   X is

-   X¹ is CH₂, O, NH, S(O)₀₋₂, 3- to 7-membered cycloalkylene or    heterocycloalkylene unsubstituted or substituted with (CH₂)₀₋₅X² or    O(CH₂)₂₋₅X², or 5- to 6-membered arylene or heteroarylene    unsubstituted or substituted with (CH₂)₀₋₅X² or O(CH₂)₂₋₅X²;-   each X² is independently Me, CO₂H, NH₂, NH(C₁-C₅ alkyl), N(C₁-C₅    alkyl)₂, SH, CHO, N(CH₂CH₂)₂N(C₁-C₃ alkyl), N(CH₂CH₂)₂NH, NHNH₂, or    C(═O)NHNH₂;-   Y is (CH₂)₄₋₆CH═CH(CH₂)₄₋₆, (CH₂)₄₋₆X¹(CH₂)₄₋₆, or    (CH₂)₂(OCH₂CH₂)₂₋₃;-   each R¹ and R² is independently H, F, Cl, Br, OH, C₁-C₃ alkyl,    O(C₁-C₃ alkyl), cyano, (CH₂)₀₋₅NH₂, or NO₂ (with both R¹ and R²    preferably being H);-   each double line    in a diazepine ring system independently represents a single bond or    a double bond;-   each R³ is H if the double line    to the N to which it is attached—i.e., with which it is    associated—is a single bond and is absent if the double line is a    double bond;-   each R⁴ is H, OH, SO₃Na, or SO₃K if the double line    to the C to which it is attached—i.e., with which it is    associated—is a single bond and is absent if the double line is a    double bond;-   A and B are independently according to formula Ia or Ib

-   -   wherein, in formula Ia    -   Y′ and Y″ are independently absent, CH₂, C═O, or CHR¹²; wherein        each R¹² is independently F, Cl, Br, or C₁-C₃ alkyl, with the        proviso that Y′ and Y″ are not both absent;    -   each G is independently C or N, with the proviso that no more        than two Gs are N; and    -   each R⁵, R⁶, R⁷, and R⁸ is independently H, C₁-C₅ alkyl,        C≡C(CH₂)₁₋₅X², OH, O(C₁-C₅ alkyl), cyano, NO₂, F, Cl, Br,        O(CH₂CH₂O)₁₋₈(C₁₋₃ alkyl), (CH₂)₀₋₅X², O(CH₂)₂₋₅X², 3- to        7-membered cycloalkyl or heterocycloalkyl unsubstituted or        substituted with (CH₂)₀₋₅X² or O(CH₂)₂₋₅X², 5- to 6-membered        aryl or heteroaryl unsubstituted or substituted with (CH₂)₀₋₅X²        or O(CH₂)₂₋₅X²,

-   -   or where a R⁵, R⁶, R⁷, or R⁸ is attached to—i.e., is associated        with—a G that is N, such R⁵, R⁶, R⁷, or R⁸ is absent;    -   and    -   wherein, in formula Ib,    -   the dotted lines indicate the optional presence of a C1-C2,        C2-C3, or C2-R¹⁰ double bond;    -   R⁹ is absent if a C1-C2, C2-C3, or C2-R¹⁰ double bond is present        and otherwise is H; and    -   R¹⁰ is H, ═O, ═CH₂, ═CH(C₁-C₅ alkyl), CH═CH(CH₂)₁₋₅X²,        C≡C(CH₂)₁₋₅X², C₁-C₅ alkyl, OH, O(C₁-C₅ alkyl), cyano, NO₂, F,        Cl, Br, O(CH₂CH₂O)₁₋₈(C₁₋₃ alkyl), (CH₂)₀₋₅X², 4- to 7-membered        aryl, heteroaryl, cycloalkyl, or heterocycloalkyl unsubstituted        or substituted with (CH₂)₀₋₅X², O(CH₂)₂₋₅X², 3- to 7-membered        cycloalkyl or heterocycloalkyl unsubstituted or substituted with        (CH₂)₀₋₅X² or O(CH₂)₂₋₅X², 5- to 6-membered aryl or heteroaryl        unsubstituted or substituted with (CH₂)₀₋₅X² or O(CH₂)₂₋₅X²;        or a pharmaceutically acceptable salt thereof.

In another embodiment, this invention provides a conjugate comprising adimer of formula (I) covalently bonded to a targeting moiety thatspecifically or preferentially binds to a chemical entity on a targetcell, which target cell preferably is a cancer cell. Preferably, thetargeting moiety is an antibody—more preferably a monoclonal antibody;even more preferably a human monoclonal antibody—and the chemical entityis a tumor associated antigen. The tumor associated antigen can be onethat is displayed on the surface of a cancer cell or one that issecreted by a cancer cell into the surrounding extracellular space.Preferably, the tumor associated antigen is one that is over-expressedby the cancer cell compared to normal cells or one that is expressed bycancer cells but not normal cells.

In another embodiment, there is provided a dimer according to formula(I) covalently bonded to a linker moiety having a reactive functionalgroup, suitable for conjugation to a targeting moiety.

In another embodiment, there is provided a method for treating a cancerin a subject suffering from such cancer, comprising administering to thesubject a therapeutically effective amount of a dimer of this inventionor a conjugate thereof with a targeting moiety. In another embodiment,there is provided the use of a dimer of this invention or a conjugatethereof with a targeting moiety for the preparation of a medicament forthe treatment of cancer in a subject suffering from such cancer. A dimerof this invention or a conjugate thereof with a targeting moiety canalso be used to inhibit the proliferation, in vitro, ex vivo, or invivo, of cancer cells. Especially, the cancer is lung, gastric, orovarian cancer.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIGS. 1A-1B, 2, 3, 4A-4B, 5, 6, 7, 8, 9, and 10 show reaction schemesfor the synthesis of dimers of this invention.

FIGS. 11, 12, 13A-13B, 14, and 15 show reaction schemes for thepreparation of dimer-linker compounds usable for the preparation ofADCs.

FIGS. 16A-16B show, in combination, the synthesis of another dimer ofthis invention.

FIG. 17 shows the activity of two ADCs of this invention against cancercells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Antibody” means whole antibodies and any antigen binding fragment(i.e., “antigen-binding portion”) or single chain variants thereof. Awhole antibody is a protein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds. Each heavychain comprises a heavy chain variable region (V_(H)) and a heavy chainconstant region comprising three domains, C_(H1), C_(H2) and C_(H3).Each light chain comprises a light chain variable region (V_(L) orV_(k)) and a light chain constant region comprising one single domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with more conserved framework regions (FRs). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged from amino-to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions contain a binding domain thatinteracts with an antigen. The constant regions may mediate the bindingof the antibody to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. An antibody is said to “specificallybind” to an antigen X if the antibody binds to antigen X with a K_(D) of5×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, more preferably6×10⁻⁹ M or less, more preferably 3×10⁻⁹ M or less, even more preferably2 ×10⁻⁹ M or less. The antibody can be chimeric, humanized, or,preferably, human. The heavy chain constant region can be engineered toaffect glycosylation type or extent, to extend anti-body half-life, toenhance or reduce interactions with effector cells or the complementsystem, or to modulate some other property. The engineering can beaccomplished by replacement, addition, or deletion of one or more aminoacids or by replacement of a domain with a domain from anotherimmunoglobulin type, or a combination of the foregoing.

“Antigen binding fragment” and “antigen binding portion” of an antibody(or simply “antibody portion” or “antibody fragment”) mean one or morefragments of an antibody that retain the ability to specifically bind toan antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody, suchas (i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fab′ fragment, which is essentially an Fabwith part of the hinge region (see, for example, Abbas et al., Cellularand Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) a Fdfragment consisting of the V_(H) and C_(H1) domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Preferred antigen binding fragments are Fab, F(ab′)₂, Fab′, Fv, and Fdfragments. Furthermore, although the two domains of the Fv fragment,V_(L) and V_(H), are encoded by separate genes, they can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv, or scFv); see,e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also encompassed within the term “antigen-binding portion” of anantibody.

An “isolated antibody” means an antibody that is substantially free ofother antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds antigen X is substantiallyfree of antibodies that specifically bind antigens other than antigenX). An isolated antibody that specifically binds antigen X may, however,have cross-reactivity to other antigens, such as antigen X moleculesfrom other species. In certain embodiments, an isolated antibodyspecifically binds to human antigen X and does not cross-react withother (non-human) antigen X antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

“Monoclonal antibody” or “monoclonal antibody composition” means apreparation of antibody molecules of single molecular composition, whichdisplays a single binding specificity and affinity for a particularepitope.

“Human antibody” means an antibody having variable regions in which boththe framework and CDR regions (and the constant region, if present) arederived from human germline immunoglobulin sequences. Human antibodiesmay include later modifications, including natural or syntheticmodifications. Human antibodies may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

“Human monoclonal antibody” means an antibody displaying a singlebinding specificity, which has variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, human monoclonal antibodies are producedby a hybridoma that includes a B cell obtained from a transgenicnonhuman animal, e.g., a transgenic mouse, having a genome comprising ahuman heavy chain transgene and a light chain transgene fused to animmortalized cell.

“Aliphatic” means a straight- or branched-chain, saturated orunsaturated, non-aromatic hydrocarbon moiety having the specified numberof carbon atoms (e.g., as in “C₃ aliphatic,” “C₁₋₅ aliphatic,” “C₁-C₅aliphatic,” or “C₁ to C₅ aliphatic,” the latter three phrases beingsynonymous for an aliphatic moiety having from 1 to 5 carbon atoms) or,where the number of carbon atoms is not explicitly specified, from 1 to4 carbon atoms (2 to 4 carbons in the instance of unsaturated aliphaticmoieties). A similar understanding is applied to the number of carbonsin other types, as in C₂₋₄ alkene, C₄-C₇ cycloaliphatic, etc. In asimilar vein, a term such as “(CH₂)₁₋₃” is to be understand as shorthadfor the subscript being 1, 2, or 3, so that such term represents CH₂,CH₂CH₂, and CH₂CH₂CH_(2.)

“Alkyl” means a saturated aliphatic moiety, with the same convention fordesignating the number of carbon atoms being applicable. By way ofillustration, C₁-C₄ alkyl (or, synonymously, C₁₋₄ alkyl) moietiesinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,isobutyl, t-butyl, 1-butyl, 2-butyl, and the like. “Alkylene” means adivalent counterpart of an alkyl group, such as CH₂CH₂, CH₂CH₂CH₂, andCH₂CH₂CH₂CH_(2.)

“Alkenyl” means an aliphatic moiety having at least one carbon-carbondouble bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkenylmoieties include, but are not limited to, ethenyl (vinyl), 2-propenyl(allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E- (or Z-)2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl) and the like.

“Alkynyl” means an aliphatic moiety having at least one carbon-carbontriple bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkynylgroups include ethynyl (acetylenyl), propargyl (prop-2-ynyl),1-propynyl, but-2-ynyl, and the like.

“Cycloaliphatic” means a saturated or unsaturated, non-aromatichydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to8 (preferably from 3 to 6) carbon atoms. “Cycloalkyl” means acycloaliphatic moiety in which each ring is saturated. “Cycloalkenyl”means a cycloaliphatic moiety in which at least one ring has at leastone carbon-carbon double bond. “Cycloalkynyl” means a cycloaliphaticmoiety in which at least one ring has at least one carbon-carbon triplebond. By way of illustration, cycloaliphatic moieties include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl.Preferred cycloaliphatic moieties are cycloalkyl ones, especiallycyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. “Cycloalkylene”means a divalent counterpart of a cycloalkyl group.

“Heterocycloaliphatic” means a cycloaliphatic moiety wherein, in atleast one ring thereof, up to three (preferably 1 to 2) carbons havebeen replaced with a heteroatom independently selected from N, O, or S,where the N and S optionally may be oxidized and the N optionally may bequaternized. Similarly, “heterocycloalkyl,” “heterocycloalkenyl,” and“heterocycloalkynyl” means a cycloalkyl, cycloalkenyl, or cycloalkynylmoiety, respectively, in which at least one ring thereof has been somodified. Exemplary heterocycloaliphatic moieties include aziridinyl,azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl,piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl,tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl,thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolanyl,tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl, thietanyl, and the like.“Heterocycloalkylene” means a divalent counterpart of a heterocycloalkylgroup.

“Alkoxy,” “aryloxy,” “alkylthio,” and “arylthio” mean —O(alkyl),—O(aryl), —S(alkyl), and —S(aryl), respectively. Examples are methoxy,phenoxy, methylthio, and phenylthio, respectively.

“Halogen” or “halo” means fluorine, chlorine, bromine or iodine.

“Aryl” means a hydrocarbon moiety having a mono-, bi-, or tricyclic ringsystem wherein each ring has from 3 to 7 carbon atoms and at least onering is aromatic. The rings in the ring system may be fused to eachother (as in naphthyl) or bonded to each other (as in biphenyl) and maybe fused or bonded to non-aromatic rings (as in indanyl orcyclohexyl-phenyl). By way of further illustration, aryl moietiesinclude, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, biphenyl, phenanthryl, anthracenyl, and acenaphthyl. “Arylene”means a divalent counterpart of an aryl group, for example1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.

“Heteroaryl” means a moiety having a mono-, bi-, or tricyclic ringsystem wherein each ring has from 3 to 7 carbon atoms and at least onering is an aromatic ring containing from 1 to 4 heteroatomsindependently selected from from N, O, or S, where the N and Soptionally may be oxidized and the N optionally may be quaternized. Suchat least one heteroatom containing aromatic ring may be fused to othertypes of rings (as in benzofuranyl or tetrahydroisoquinolyl) or directlybonded to other types of rings (as in phenylpyridyl or2-cyclopentylpyridyl). By way of further illustration, heteroarylmoieties include pyrrolyl, furanyl, thiophenyl (thienyl), imidazolyl,pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,tetrazolyl, pyridyl, N-oxopyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,quinolinyl, isoquinolynyl, quinazolinyl, cinnolinyl, quinozalinyl,naphthyridinyl, benzofuranyl, indolyl, benzothiophenyl, oxadiazolyl,thiadiazolyl, phenothiazolyl, benzimidazolyl, benzotriazolyl,dibenzofuranyl, carbazolyl, dibenzothiophenyl, acridinyl, and the like.“Heteroarylene” means a divalent counterpart of a heteroaryl group.

Where it is indicated that a moiety may be substituted, such as by useof “unsubstituted or substituted” or “optionally substituted” phrasingas in “unsubstituted or substituted C₁-C₅ alkyl” or “optionallysubstituted heteroaryl,” such moiety may have one or more independentlyselected substituents, preferably one to five in number, more preferablyone or two in number. Substituents and substitution patterns can beselected by one of ordinary skill in the art, having regard for themoiety to which the substituent is attached, to provide compounds thatare chemically stable and that can be synthesized by techniques known inthe art as well as the methods set forth herein.

“Arylalkyl,” (heterocycloaliphatic)alkyl,” “arylalkenyl,” “arylalkynyl,”“biarylalkyl,” and the like mean an alkyl, alkenyl, or alkynyl moiety,as the case may be, substituted with an aryl, heterocycloaliphatic,biaryl, etc., moiety, as the case may be, with the open (unsatisfied)valence at the alkyl, alkenyl, or alkynyl moiety, for example as inbenzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like.Conversely, “alkylaryl,” “alkenylcycloalkyl,” and the like mean an aryl,cycloalkyl, etc., moiety, as the case may be, substituted with an alkyl,alkenyl, etc., moiety, as the case may be, for example as inmethylphenyl (tolyl) or allylcyclohexyl. “Hydroxyalkyl,” “haloalkyl,”“alkylaryl,” “cyanoaryl,” and the like mean an alkyl, aryl, etc.,moiety, as the case may be, substituted with one or more of theidentified substituent (hydroxyl, halo, etc., as the case may be).

For example, permissible substituents include, but are not limited to,alkyl (especially methyl or ethyl), alkenyl (especially allyl), alkynyl,aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especiallyfluoro), haloalkyl (especially trifluoromethyl), hydroxyl, hydroxyalkyl(especially hydroxyethyl), cyano, nitro, alkoxy, —O(hydroxyalkyl),—O(haloalkyl) (especially —OCF3), —O(cycloalkyl), —O(heterocycloalkyl),—O(aryl), alkylthio, arylthio, ═O, ═NH, ═N(alkyl), ═NOH, ═NO(alkyl),—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl),—SO₂N(alkyl)₂, and the like.

Where the moiety being substituted is an aliphatic moiety, preferredsubstituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic,halo, hydroxyl, cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl),—O(cycloalkyl), —O(heterocycloalkyl), —O(aryl), alkylthio, arylthio, ═O,═NH, ═N(alkyl), ═NOH, ═NO(alkyl), —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(═O)alkyl, —S(cycloalkyl), —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are halo, hydroxyl, cyano,nitro, alkoxy, —O(aryl), ═O, ═NOH, ═NO(alkyl), —OC(═O)(alkyl),—OC(═O)O(alkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂, azido,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are phenyl, cyano, halo, hydroxyl, nitro,C₁-C₄alkyoxy, O(C₂-C₄ alkylene)OH, and O(C₂-C₄ alkylene)halo.

Where the moiety being substituted is a cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl moiety, preferred substituentsare alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl,cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl), —O(aryl),—O(cycloalkyl), —O(heterocycloalkyl), alkylthio, arylthio,—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are alkyl, alkenyl, halo,haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy,—O(hydroxyalkyl), —C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH,—C(═O)O(alkyl), —C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl),—C(═O)N(alkyl)₂, —OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are C₁-C₄ alkyl, cyano, nitro, halo, andC₁-C₄alkoxy.

Where a range is stated, as in “C₁-C₅ alkyl” or “5 to 10%,” such rangeincludes the end points of the range, as in C₁ and C₅ in the firstinstance and 5% and 10% in the second instance.

Unless particular stereoisomers are specifically indicated (e.g., by abolded or dashed bond at a relevant stereocenter in a structuralformula, by depiction of a double bond as having E or Z configuration ina structural formula, or by use stereochemistry-designatingnomenclature), all stereoisomers are included within the scope of theinvention, as pure compounds as well as mixtures thereof. Unlessotherwise indicated, individual enantiomers, diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythis invention.

Those skilled in the art will appreciate that compounds may havetautomeric forms (e.g., keto and enol forms), resonance forms, andzwitterionic forms that are equivalent to those depicted in thestructural formulae used herein and that the structural formulaeencompass such tautomeric, resonance, or zwitterionic forms.

“Pharmaceutically acceptable ester” means an ester that hydrolyzes invivo (for example in the human body) to produce the parent compound or asalt thereof or has per se activity similar to that of the parentcompound. Suitable esters include C₁-C₅ alkyl, C₂-C₅ alkenyl or C₂-C₅alkynyl esters, especially methyl, ethyl or n-propyl.

“Pharmaceutically acceptable salt” means a salt of a compound suitablefor pharmaceutical formulation. Where a compound has one or more basicgroups, the salt can be an acid addition salt, such as a sulfate,hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate,pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate,methyl-sulfate, fumarate, benzoate, succinate, mesylate, lactobionate,suberate, tosylate, and the like. Where a compound has one or moreacidic groups, the salt can be a salt such as a calcium salt, potassiumsalt, magnesium salt, meglumine salt, ammonium salt, zinc salt,piperazine salt, tromethamine salt, lithium salt, choline salt,diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodiumsalt, tetramethylammonium salt, and the like. Polymorphic crystallineforms and solvates are also encompassed within the scope of thisinvention.

In the formulae of this specification, a wavy line

transverse to a bond or an asterisk (*) at the end of the bond denotes acovalent attachment site. For instance, a statement that R is

in the formula

refers to

In the formulae of this specification, a bond traversing an aromaticring between two carbons thereof means that the group attached to thebond may be located at any of the available positions of the aromaticring. By way of illustration, the formula

represents

Preferably, in formula I, X is a C7/C7′ bridge that is 3 or 5 atoms longand Y is a C8/C8′ bridge that is 7, 8, 9, 10, 11, or 12 atoms long,where the atoms in each bridge are selected from C, O, N, and S,especially where all the bridge atoms are C or all are C except for oneO, N, or S. More preferably, where X is 3 atoms long, Y is 7, 8, 9, or10 atoms long (even more preferably 8 to 10 atoms long) and where X is 5atoms long, Y is 11 or 12 atoms long.

In a preferred embodiment, in formula I, X is (CH₂)3 or (CH₂)₅.

In another preferred embodiment, in formulae I, IIa, IIb, or IIc (thelatter three formulae shown hereinbelow), Y is (CH₂)₇₋₁₂, morepreferably (CH₂)₈.

In formula I, preferred combinations of X and Y are X equals (CH₂)₃while Y equals (CH₂)₈₋₁₀ and X equals (CH₂)₅ while Y equals (CH₂)₁₁₋₁₂.A more preferred combination is X equals (CH₂)3 and Y equals (CH₂)₈.

In another preferred embodiment, in formulae I, IIa, IIb, or IIc, Y is(CH₂)₂(OCH₂CH₂)₂₋₃, especially (CH₂)₂(OCH₂CH₂)₂.

In another preferred embodiment, in formulae I, IIa, IIb, or IIc, Y is(CH₂)₄NH(CH₂)₄ or (CH₂)₄N(CH₂(p-C₆H₄NH₂))(CH₂)₄.

In the formulae where it appears, X² preferably is NH₂, SH, or CO₂H.

Preferably, where X in formula I is

and X¹ is O or NH, then X is

In a preferred embodiment, dimers of this invention have a structurerepresented by formula IIa, wherein x is 3 or 5 (preferably 3); Y is(CH₂)₇₋₁₂, (CH₂)₂(OCH₂CH₂)₁₋₃, (CH₂)₂₋₄NH(CH₂)₂₋₄ or(CH₂)₂₋₄NH((CH₂)₀₋₁phenyl)(CH₂)₂₋₄ where the phenyl group is optionallysubstituted with NH₂; A is A1, A2, or A3 as depicted below, and B is B1,B2, or B3 as depicted below. Representative species are listed in Table1.

TABLE 1 Examples of Dimers According to Formula IIa Dimer x Y A B IIa-13 (CH₂)₇ A1 B1 IIa-2 3 (CH₂)₈ A1 A1 IIa-3 3 (CH₂)₉ A1 A1 IIa-4 3 (CH₂)₁₀A1 B1 IIa-5 3 (CH₂)₁₁ A1 A1 IIa-6 3 (CH₂)₁₂ A1 A1 IIa-7 5 (CH₂)₁₁ A1 B1IIa-8 5 (CH₂)₁₂ A1 A1 IIa-9 3

A1 A1 IIa-10 3

A2 B2 IIa-11 3 (CH₂)₁₀ A3 B3 IIa-12 3 (CH₂)₈ A2 B2 IIa-13 3(CH₂)₂(OCH₂CH₂)₂ A2 B2 IIa-14 3 (CH₂)₄NH(CH₂)₄ A2 B2

In another preferred embodiment, dimers of this invention have astructure represented by formula IIb, where x is 3 or 5 (preferably 3),each Y′ is independently absent or CH₂, Y is (CH₂)₇₋₁₂,(CH₂)₂(OCH₂CH₂)₁₋₃, (CH₂)₂₋₄NH(CH₂)₂₋₄ or(CH₂)₂₋₄NH((CH₂)₀₋₁phenyl)(CH₂)₂₋₄ where the phenyl group is optionallysubstituted with NH₂; and R⁴⁰ and R⁴¹ are independently H, Cl, Br, OH,O(C₁₋₃ alkyl), NH₂, or C₁₋₃ alkyl. Representative species are listed inTable 2.

TABLE 2 Examples of Dimers According to Formula IIb Dimer x Y′ Y R⁴⁰ R⁴¹IIb-1 3 CH₂ (CH₂)₈ H H IIb-2 3 CH₂ (CH₂)₁₀ H H IIb-3 3 absent (CH₂)₁₀ HH IIb-4 3 CH₂ (CH₂)₂(OCH₂CH₂)₂ H H IIb-5 3 CH₂ (CH₂)₄NH(CH₂)₄ H H IIb-63 CH₂ (CH₂)₈ H NH₂ IIb-7 3 CH₂ (CH₂)₂(OCH₂CH₂)₂ H NH₂ IIb-8 3 CH₂

H H

In another preferred embodiment, dimers of this invention have astructure represented by formula IIc, where x is 3 or 5 (preferably 3),Y is (CH₂)₇₋₁₂, (CH₂)₂(OCH₂CH₂)₁₋₃, (CH₂)₂₋₄NH(CH₂)₂₋₄ or(CH₂)₂₋₄NH((CH₂)₀₋₁phenyl)(CH₂)₂₋₄ where the phenyl group is optionallysubstituted with NH₂; and R⁴² and R⁴³ are independently H, OMe, NH₂,OCH₂CH₂OMe, N(CH₂CH₂)O, N(CH₂CH₂)NMe, or N(CH₂CH₂)NH. Representativespecies are listed in Table 3.

TABLE 3 Examples of Dimers According to Formula IIc Dimer x Y R⁴² R⁴³IIc-1 3 (CH₂)₁₀ H H IIc-2 3 (CH₂)₁₂ OMe OMe IIc-3 3 (CH₂)₁₂ OCH₂CH₂OMeOCH₂CH₂OMe IIc-4 3 (CH₂)₁₂

IIc-5 3 (CH₂)₁₂

IIc-6 3 (CH₂)₂(OCH₂CH₂)₂ H H IIc-7 3 (CH₂)₈ OMe OMe IIc-8 3 (CH₂)₈

IIc-9 3 (CH₂)₈

IIc-10 3 (CH₂)₈

NH₂ IIc-11 3 (CH₂)₈ NH₂ OMe

Preferably, dimers of this invention are selected from the groupconsisting of dimer IIb-6, IIc-8, IIc-9, IIc-10, IIc-11, IId-1, IId-2,and IId-3:

Conjugates General

Dimers of this invention can be used as therapeutic agents per se, butpreferably are used as conjugates with a targeting moiety thatspecifically or preferentially binds to a chemical entity on a cancercell. Preferably, the targeting moiety is an antibody or antigen bindingportion thereof and the chemical entity is a tumor associated antigen.

Thus, another embodiment of this invention is a conjugate comprisingdimer of this invention and a ligand, represented by formula (II)

[D(X^(D))_(a)(C)_(c)(X^(Z))_(b)]_(m)Z   (II)

where Z is a ligand, D is a dimer of this invention, and—(X^(D))_(a)C(X^(Z))_(b)— are collectively referred to as a “linkermoiety” or “linker” because they link Z and D. Within the linker, C is acleavable group designed to be cleaved at or near the site of intendedbiological action of dimer D; X^(D) and X^(Z) are referred to as spacermoieties (or “spacers”) because they space apart D and C and C and Z,respectively; subscripts a, b, and c are independently 0 or 1 (that is,the presence of X^(D), X^(Z) and C are optional). Subscript m is 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 (preferably 1, 2, 3, or 4). D, X^(D), C,X^(Z) and Z are more fully described hereinbelow.

Ligand Z—for example an antibody—performs a targeting function. Bybinding to a target tissue or cell where its antigen or receptor islocated, ligand Z directs the conjugate there. (When ligand Z is anantibody, the conjugate is sometimes referred to as antibody-drugconjugate (ADC) or an immunoconjugate. Preferably, the target tissue orcell is a cancer tissue or cell and the antigen or receptor is atumor-associated antigen, that is, an antigen that is uniquely expressedby cancerous cells or is overexpressed by cancer cells, compared tonon-cancerous cells. Cleavage of group C at the target tissue or cellreleases dimer D to exert its cytotoxic effect locally. In someinstances, the conjugate is internalized into a target cell byendocytosis and cleavage takes place within the target cell. In thismanner, precise delivery of dimer D is achieved at the site of intendedaction, reducing the dosage needed. Also, dimer D is normallybiologically inactive (or significantly less active) in its conjugatedstate, thereby reducing undesired toxicity against non-target tissue orcells. As anticancer drugs are often highly toxic to cells in general,this is an important consideration.

As reflected by the subscript m, each molecule of ligand Z can conjugatewith more than one dimer D, depending on the number of sites ligand Zhas available for conjugation and the experimental conditions employed.Those skilled in the art will appreciate that, while each individualmolecule of ligand Z is conjugated to an integer number of dimers D, apreparation of the conjugate may analyze for a non-integer ratio ofdimers D to ligand Z, reflecting a statistical average. This ratio isreferred to as the substitution ratio (SR) or, synonymously, thedrug-antibody ratio (DAR).

Ligand Z

Preferably, ligand Z is an antibody. For convenience and brevity and notby way of limitation, the detailed subsequent discussion herein aboutthe conjugation of ligand Z is written in the context of its being anantibody, but those skilled in the art will understand that other typesof ligand Z can be conjugated, mutatis mutandis. For example, conjugateswith folic acid as the ligand can target cells having the folatereceptor on their surfaces (Leamon et al., Cancer Res. 2008, 68 (23),9839). For the same reason, the detailed discussion below is primarilywritten in terms of a 1:1 ratio of antibody Z to analog D (m=1).

Preferably, ligand Z is an antibody against a tumor associated antigen,allowing the selective targeting of cancer cells. Examples of suchantigens include: mesothelin, prostate specific membrane antigen (PSMA),CD19, CD22, CD30, CD70, B7H3, B7H4 (also known as O8E), protein tyrosinekinase 7 (PTK7), glypican-3, RG1, fucosyl-GM1, CTLA-4, and CD44. Theantibody can be animal (e.g., murine), chimeric, humanized, or,preferably, human. The antibody preferably is monoclonal, especially amonoclonal human antibody. The preparation of human monoclonalantibodies against some of the aforementioned antigens is disclosed inKorman et al., U.S. Pat. No. 8,609,816 B2 (2013; B7H4, also known as08E; in particular antibodies 2A7, 1G11, and 2F9); Rao-Naik et al.,8,097,703 B2 (2012; CD19; in particular antibodies 5G7, 13F1, 46E8,21D4, 21D4a, 47G4, 27F3, and 3C10); King et al., U.S. Pat. No. 8,481,683B2 (2013; CD22; in particular antibodies 12C5, 19A3, 16F7, and 23C6);Keler et al., U.S. Pat. No. 7,387,776 B2 (2008; CD30; in particularantibodies 5F11, 2H9, and 17G1); Terrett et al., U.S. Pat. No. 8,124,738B2 (2012; CD70; in particular antibodies 2H5, 10B4, 8B5, 18E7, and69A7); Korman et al., U.S. Pat. No. 6,984,720 B1 (2006; CTLA-4; inparticular antibodies 10D1, 4B6, and 1E2); Vistica et al., U.S. Pat. No.8,383,118 B2 (2013, fucosyl-GM1, in particular antibodies 5B1, 5B1a,7D4, 7E4, 13B8, and 18D5) Korman et al., U.S. Pat. No. 8,008,449 B2(2011; PD-1; in particular antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3,and 5F4); Huang et al., US 2009/0297438 A1 (2009; PSMA. in particularantibodies 1C3, 2A10, 2F5, 2C6); Cardarelli et al., U.S. Pat. No.7,875,278 B2 (2011; PSMA; in particular antibodies 4A3, 7F12, 8C12,8A11, 16F9, 2A10, 2C6, 2F5, and 1C3); Terrett et al., U.S. Pat. No.8,222,375 B2 (2012; PTK7; in particular antibodies 3G8, 4D5, 12C6,12C6a, and 7C8); Terrett et al., U.S. Pat. No. 8,680,247 B2 (2014;glypican-3; in particular antibodies 4A6, 11E7, and 16D10); Harkins etal., U.S. Pat. No. 7,335,748 B2(2008; RG1; in particular antibodies A,B, C, and D); Terrett et al., U.S. Pat. No. 8,268,970 B2 (2012;mesothelin; in particular antibodies 3C10, 6A4, and 7B1); Xu et al., US2010/0092484 A1 (2010; CD44; in particular antibodies 14G9.B8.B4,2D1.A3.D12, and 1A9.A6.B9); Deshpande et al., U.S. Pat. No. 8,258,266 B2(2012; IP10; in particular antibodies 1D4, 1E1, 2G1, 3C4, 6A5, 6A8,7C10, 8F6, 10A12, 10A12S, and 13C4); Kuhne et al., Us 8,450,464 B2(2013; CXCR4; in particular antibodies F7, F9, D1, and E2); and Kormanet al., U.S. Pat. No. 7,943,743 B2 (2011; PD-L1; in particularantibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B 12, 7H1, 11E6, 12B7, and13G4); the disclosures of which are incorporated herein by reference.Each of the aforementioned antibodies can be used in an ADC with d dimerof this invention.

Ligand Z can also be an antibody fragment or antibody mimetic, such asan affibody, a domain antibody (dAb), a nanobody, a unibody, a DARPin,an anticalin, a versabody, a duocalin, a lipocalin, or an avimer.

Any one of several different reactive groups on ligand Z can be aconjugation site, including ε-amino groups in lysine residues, pendantcarbohydrate moieties, carboxylic acid groups, disulfide groups, andthiol groups. Each type of reactive group represents a trade-off, havingsome advantages and some disadvantages. For reviews on antibody reactivegroups suitable for conjugation, see, e.g., Garnett, Adv. Drug DeliveryRev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology &Therapeutics 83 (1999), 67-123, the disclosures of which areincorporated herein by reference.

In one embodiment, ligand Z is conjugated via a lysine ε-amino group.Most antibodies have multiple lysine ε-amino groups, which can beconjugated via amide, urea, thiourea, or carbamate bonds usingtechniques known in the art. However, it is difficult to control whichand how many ε-amino groups react, leading to potential batch-to-batchvariability in conjugate preparations. Also, conjugation may causeneutralization of a protonated C-amino group important for maintainingthe antibody's native conformation or may take place at a lysine near orat the antigen binding site, neither being a desirable occurrence.

In another embodiment, ligand Z can be conjugated via a carbohydrateside chain, as many antibodies are glycosylated. The carbohydrate sidechain can be oxidized with periodate to generate aldehyde groups, whichin turn can be reacted with amines to form an imine group, such as in asemicarbazone, oxime, or hydrazone. If desired, the imine group can beconverted to a more stable amine group by reduction with sodiumcyanoborohydride. For additional disclosures on conjugation viacarbohydrate side chains, see, e.g., Rodwell et al., Proc. Nat'l Acad.Sci. USA 83, 2632-2636 (1986); the disclosure of which is incorporatedherein by reference. As with lysine ε-amino groups, there are concernsregarding reproducibility of the location of the conjugation site(s) andstoichiometry.

In yet another embodiment, ligand Z can be conjugated via a carboxylicacid group. In one embodiment, a terminal carboxylic acid group isfunctionalized to generate a carbohydrazide, which is then reacted withan aldehyde-bearing conjugation moiety. See Fisch et al., BioconjugateChemistry 1992, 3, 147-153 .

In yet another embodiment, antibody Z can be conjugated via a disulfidegroup bridging a cysteine residue on antibody Z and a sulfur on theother portion of the conjugate. Some antibodies lack free thiol(sulfhydryl) groups but have disulfide groups, for example in the hingeregion. In such case, free thiol groups can be generated by reduction ofnative disulfide groups. The thiol groups so generated can then be usedfor conjugation. See, e.g., Packard et al., Biochemistry 1986, 25,3548-3552; King et al., Cancer Res. 54, 6176-6185 (1994); and Doroninaet al., Nature Biotechnol. 21(7), 778-784 (2003); the disclosures ofwhich are incorporated herein by reference. Again, there are concernsregarding conjugation site location and stoichiometry and the possibledisruption of antibody native conformation.

A number of methods are known for introducing free thiol groups intoantibodies without breaking native disulfide bonds, which methods can bepracticed with a ligand Z of this invention. Depending on the methodemployed, it may be possible to introduce a predictable number of freesulfhydryls at predetermined locations. In one approach, mutatedantibodies are prepared in which a cysteine is substituted for anotheramino acid. See, for example, Eigenbrot et al., U.S. Pat. No. 7,521,541B2 (2009); Chilkoti et al., Bioconjugate Chem. 1994, 5, 504-507;Urnovitz et al., U.S. Pat. No. 4,698,420 (1987); Stimmel et al., J.Biol. Chem., 275 (39), 30445-30450 (2000); Bam et al., U.S. Pat. No.7,311,902 B2 (2007); Kuan et al., J. Biol. Chem., 269 (10), 7610-7618(1994); Poon et al., J. Biol. Chem., 270 (15), 8571-8577 (1995). Inanother approach, an extra cysteine is added to the C-terminus. See,e.g. Cumber et al., J. Immunol., 149, 120-126 (1992); King et al, CancerRes., 54, 6176-6185 (1994); Li et al., Bioconjugate Chem., 13, 985-995(2002); Yang et al., Protein Engineering, 16, 761-770 (2003); andOlafson et al., Protein Engineering Design & Selection, 17, 21-27(2004). A preferred method for introducing free cysteines is that taughtby Liu et al., WO 2009/026274 A1, in which a cysteine bearing amino acidsequence is added to the C-terminus of the heavy chain of an antibody.This method introduces a known number of cysteine residues (one perheavy chain) at a known location awat from the antigen binding site. Thedisclosures of the documents cited in this paragraph are allincorporated herein by reference.

In yet another embodiment, lysine ε-amino groups can be modified withreagents such as 2-iminothiolane orN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), converting anε-amino group into a thiol or disulfide group—creating a cysteinesurrogate, as it were. However, this method suffers from the sameconjugation location and stoichiometry limitations associated withε-amino groups proper.

Linker Components

As noted above, the linker portion of a conjugate of this inventioncomprises up to three elements: a cleavable group C and optional spacersX^(Z) and X^(D).

Cleavable group C is a group cleavable under physiological conditions,preferably selected such that it is relatively stable while theconjugate is in general circulation in the blood plasma, but is readilycleaved once the conjugate reaches its site of intended action, that is,near, at, or within the target cell. Preferably, the conjugate isinternalized by a target cell upon binding of antibody Z to an antigendisplayed on the surface of the target cell. Subsequently, cleavage ofgroup C occurs in a vesicular body of the target cell (an earlyendosome, a late endosome, or, especially, a lysosome).

In one embodiment, group C is a pH sensitive group. The pH in bloodplasma is slightly above neutral, while the pH inside a lysosome isacidic, circa 5. Thus, a group C whose cleavage is acid catalyzed willcleave at a rate several orders of magnitude faster inside a lysosomethan in the blood plasma rate. Examples of suitable acid-sensitivegroups include cis-aconityl amides and hydrazones, as described in Shenet al., U.S. Pat. No. 4,631,190 (1986); Shen et al., U.S. Pat. No.5,144,011 (1992); Shen et al., Biochem. Biophys. Res. Commun. 102,1048-1054 (1981) and Yang et al., Proc. Natl Acad. Sci (USA), 85,1189-1193 (1988); the disclosures of which are incorporated herein byreference.

In another embodiment, group C is a disulfide. Disulfides can be cleavedby a thiol-disulfide exchange mechanism, at a rate dependent on theambient thiol concentration. As the intracellular concentration ofglutathione and other thiols is higher than their serum concentrations,the cleavage rate of a disulfide will be higher intracellularly.Further, the rate of thiol-disulfide exchange can be modulated byadjustment of the steric and electronic characteristics of the disulfide(e.g., an alkyl-aryl disulfide versus an alkyl-alkyl disulfide;substitution on the aryl ring, etc.), enabling the design of disulfidelinkages that have enhanced serum stability or a particular cleavagerate. For additional disclosures relating to disulfide cleavable groupsin conjugates, see, e.g., Thorpe et al., Cancer Res. 48, 6396-6403(1988); Santi et al., U.S. Pat. No. 7,541,530 B2 (2009); Ng et al., U.S.Pat. No. 6,989,452 B2 (2006); Ng et al., WO 2002/096910 A1; Boyd et al.,U.S. Pat. No. 7,691,962 B2; and Sufi et al., US 2010/0145036 A1; thedisclosures of which are incorporated herein by reference.

A preferred cleavable group is a peptide that is cleaved selectively bya protease inside the target cell, as opposed to by a protease in theserum. Typically, a cleavable peptide group comprises from 1 to 20 aminoacids, preferably from 1 to 6 amino acids, more preferably from 1 to 3amino acids. The amino acid(s) can be natural and/or non-natural α-aminoacids. Natural amino acids are those encoded by the genetic code, aswell as amino acids derived therefrom, e.g., hydroxyproline,γ-carboxyglutamate, citrulline, and O-phosphoserine. In this context,the term “amino acid” also includes amino acid analogs and mimetics.Analogs are compounds having the same general H₂N(R)CHCO₂H structure ofa natural amino acid, except that the R group is not one found among thenatural amino acids. Examples of analogs include homoserine, norleucine,methionine-sulfoxide, and methionine methyl sulfonium. An amino acidmimetic is a compound that has a structure different from the generalchemical structure of an α-amino acid but functions in a manner similarto one. The amino acid can be of the “L” stereochemistry of thegenetically encoded amino acids, as well as of the enantiomeric “D”stereochemistry.

Preferably, group C contains an amino acid sequence that is a cleavagerecognition sequence for a protease. Many cleavage recognition sequencesare known in the art. See, e.g., Matayoshi et al. Science 247: 954(1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth.Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994);Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol.244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); thedisclosures of which are incorporated herein by reference.

For conjugates that are not intended to be internalized by a cell, agroup C can be chosen such that it is cleaved by a protease present inthe extracellular matrix in the vicinity of the target tissue, e.g., aprotease released by nearby dying cells or a tumor-associated protease.Exemplary extracellular tumor-associated proteases are matrixmetalloproteases (MMP), thimet oligopeptidase (TOP) and CD10.

For conjugates that are designed to be internalized by a cell, group Cpreferably comprises an amino acid sequence selected for cleavage by anendosomal or lysosomal protease, especially the latter. Non-limitingexamples of such proteases include cathepsins B, C, D, H, L and S,especially cathepsin B. Cathepsin B preferentially cleaves peptides at asequence -AA²-AA¹- where AA¹ is a basic or strongly hydrogen bondingamino acid (such as lysine, arginine, or citrulline) and AA² is ahydrophobic amino acid (such as phenylalanine, valine, alanine, leucine,or isoleucine), for example Val-Cit (where Cit denotes citrulline) orVal-Lys. (Herein, amino acid sequences are written in the N-to-Cdirection, as in H₂N-AA²-AA¹-CO₂H, unless the context clearly indicatesotherwise.) Lys-Val-Ala, Asp-Val-Ala, Val-Ala, Lys-Val-Cit, andAsp-Val-Cit are also substrate peptide motifs for cathpsin B, althoughin some instances the cleavage rate may be slower. For additionalinformation regarding cathepsin-cleavable groups, see Dubowchik et al.,Biorg. Med. Chem. Lett. 8, 3341-3346 (1998); Dubowchik et al., Bioorg.Med. Chem. Lett., 8 3347-3352 (1998); and Dubowchik et al., BioconjugateChem. 13, 855-869 (2002); the disclosures of which are incorporated byreference. Another enzyme that can be utilized for cleaving peptidyllinkers is legumain, a lysosomal cysteine protease that preferentiallycleaves at Ala-Ala-Asn.

In one embodiment, Group C is a peptide comprising a two-amino acidsequence -AA²-AA¹- wherein AA¹ is lysine, arginine, or citrulline andAA² is phenylalanine, valine, alanine, leucine or isoleucine. In anotherembodiment, C consists of a sequence of one to three amino acids,selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys,Cit, Ser, and Glu.

The preparation and design of cleavable groups C consisting of a singleamino acid is disclosed in Chen et al., U.S. Pat. No. 8,664,407 B2(2014), the disclosure of which is incorporated herein by reference.

Group C can also be a photocleavable one, for example a nitrobenzylether that is cleaved upon exposure to light.

Group C can be bonded directly to antibody Z or analog D; i.e. spacersX^(Z) and X^(D), as the case may be, can be absent. For example, ifgroup C is a disulfide, one of the two sulfurs can be a cysteine residueor its surrogate on antibody Z. Or, group C can be a hydrazone bonded toan aldehyde on a carbohydrate side chain of the antibody. Or, group Ccan be a peptide bond formed with a lysine ε-amino group of antibody Z.In a preferred embodiment, dimer D is directly bonded to group C via apeptidyl bond to a carboxyl or amine group in dimer D.

When present, spacer X^(z) provides spatial separation between group Cand antibody Z, lest the former sterically interfere with antigenbinding by latter or the latter sterically interfere with cleavage ofthe former. Further, spacer X^(z) can be used to confer increasedsolubility or decreased aggregation properties to conjugates. A spacerX^(z) can comprise one or more modular segments, which can be assembledin any number of combinations. Examples of suitable segments for aspacer X^(z) are:

and combinations thereof, where the subscript g is 0 or 1 and thesubscript h is 1 to 24, preferably 2 to 4. These segments can becombined, such as illustrated below:

Spacer X^(D), if present, provides spatial separation between group Cand dimer D, lest the latter interfere sterically or electronically withcleavage of the former. Spacer X^(D) also can serve to introduceadditional molecular mass and chemical functionality into a conjugate.Generally, the additional mass and functionality will affect the serumhalf-life and other properties of the conjugate. Thus, through judiciousselection of spacer groups, the serum half-live of a conjugate can bemodulated. Spacer X^(D) also can be assembled from modular segments, asdescribed above in the context of spacer X^(z).

Spacers X^(Z) and/or X^(D), where present, preferably provide a linearseparation of from 4 to 25 atoms, more preferably from 4 to 20 atoms,between Z and C or D and C, respectively.

The linker can perform other functions in addition to covalently linkingthe antibody and the drug. For instance, the linker can containpoly(ethylene glycol) (PEG) groups, which enhance solubility eitherduring the performance the conjugation chemistry or in the final ADCproduct. Where a PEG group is present, it may be incorporated intoeither spacer X^(Z) of X^(D), or both. The number of repeat units in aPEG group can be from 2 to 20, preferably between 4 and 10.

Either spacer X^(Z) or X^(D), or both, can comprise a self-immolatingmoiety. A self-immolating moiety is a moiety that (1) is bonded to groupC and either antibody Z or dimer D and (2) has a structure such thatcleavage from group C initiates a reaction sequence resulting in theself-immolating moiety disbonding itself from antibody Z or dimer D, asthe case may be. In other words, reaction at a site distal from antibodyZ or dimer D (cleavage from group C) causes the X^(Z)-Z or the X^(D)-Dbond to rupture as well. The presence of a self-immolating moiety isdesirable in the case of spacer X^(D) because, if, after cleavage of theconjugate, spacer X^(D) or a portion thereof were to remain attached todimer D, the biological activity of the latter may be impaired. The useof a self-immolating moiety is especially desirable where cleavablegroup C is a polypeptide, in which instance the self-immolating moietytypically is located adjacent thereto.

Exemplary self-immolating moieties (i)-(v) bonded to a hydroxyl or aminogroup on a partner molecule D are shown below:

The self-immolating moiety is the structure between dotted lines a andb, with adjacent structural features shown to provide context.Self-immolating moieties (i) and (v) are bonded to a dimer D—NH₂ (i.e.,dimer D is conjugated via an amino group), while self-immolatingmoieties (ii), (iii), and (iv) are bonded to a dimer D-OH (i.e., dimer Dis conjugated via a hydroxyl or carboxyl group). Cleavage of the amidebond at dotted line b (e.g., by a peptidase) releases the amide nitrogenas an amine nitrogen, initiating a reaction sequence that results in thecleavage of the bond at dotted line a and the consequent release of D-OHor D—NH₂, as the case may be. Alternatively, the cleavage that triggersthe self-immolating reaction can be by a different type of enzyme, forexample by a β-glucuronidase, as in the instance of structure (vi). Foradditional disclosures regarding self-immolating moieties, see Carl etal., J. Med. Chem., 24 (3), 479-480 (1981); Carl et al., WO 81/01145(1981); Dubowchik et al., Pharmacology & Therapeutics, 83, 67-123(1999); Firestone et al., U.S. Pat. No. 6,214,345 B1 (2001); Toki etal., J. Org. Chem. 67, 1866-1872 (2002); Doronina et al., NatureBiotechnology 21 (7), 778-784 (2003) (erratum, p. 941); Boyd et al.,U.S. Pat. No. 7,691,962 B2; Boyd et al., US 2008/0279868 A1; Sufi etal., WO 2008/083312 A2; Feng, U.S. Pat. No. 7,375,078 B2; Jeffrey etal., U.S. Pat. No. 8039,273; and Senter et al., US 2003/0096743 A1; thedisclosures of which are incorporated by reference. A preferredself-immolating group is p-aminobenzyl oxycarbonyl (PABC) group, asshown in structure (i).

In another embodiment, an antibody targeting moiety and the dimer D arelinked by a non-cleavable linker, i.e., element C is absent. Degradationof the antibody eventually reduces the linker to a small appended moietythat does not interfere with the biological activity of dimer D.

Conjugation Techniques

Conjugates of this invention preferably are made by first preparing acompound comprising an analog of this invention (represented by D in theformulae below) and linker (X^(D))_(a)(C)_(c)(X^(z))_(b) (where X^(D),C, X^(Z), a, b, and c are as defined for formula (II)) to form ananalog-linker composition represented by formula (III):

D-(X^(D))_(a)(C)_(c)(X^(Z))_(b)-R³¹   (III)

where R³¹ is a functional group suitable for reacting with acomplementary functional group on antibody Z to form the conjugate.Examples of suitable groups R³¹ include amino, azide, cyclooctyne,

where R³² is Cl, Br, F, mesylate, or tosylate and R³³ is Cl, Br, I, F,OH, —O—N-succinimidyl, —O-(4-nitrophenyl), —O-pentafluorophenyl, or—O-tetrafluorophenyl. Chemistry generally usable for the preparation ofsuitable moieties D-(X^(D))_(a)C(X^(Z))_(b)R³¹ is disclosed in Ng etal., U.S. Pat. No. 7,087,600 B2 (2006); Ng et al., U.S. Pat. No.6,989,452 B2 (2006); Ng et al., U.S. Pat. No. 7,129,261 B2 (2006); Ng etal., WO 02/096910 A1; Boyd et al., U.S. Pat. No. 7,691,962 B2; Chen etal., U.S. Pat. No. 7,517,903 B2 (2009); Gangwar et al., U.S. Pat. No.7,714,016 B2 (2010); Boyd et al., US 2008/0279868 A1; Gangwar et al.,U.S. Pat. No. 7,847,105 B2 (2010); Gangwar et al., U.S. Pat. No.7,968,586 B2 (2011); Sufi et al., US 2010/0145036 A1; and Chen et al.,US 2010/0113476 A1; the disclosures of which are incorporated herein byreference.

Preferably reactive functional group —R³¹ is —NH₂, —OH, —CO₂H, —SH,maleimido, cyclooctyne, azido (—N3), hydroxylamino (—ONH₂) orN-hydroxysuccinimido. Especially preferred functional groups —R³¹ are:

An —OH group can be esterified with a carboxy group on the antibody, forexample, on an aspartic or glutamic acid side chain.

A —CO₂H group can be esterified with a —OH group or amidated with anamino group (for example on a lysine side chain) on the antibody.

An N-hydroxysuccinimide group is functionally an activated carboxylgroup and can conveniently be amidated by reaction with an amino group(e.g., from lysine).

A maleimide group can be conjugated with an —SH group on the antibody(e.g., from cysteine or from the chemical modification of the antibodyto introduce a sulfhydryl functionality), in a Michael additionreaction.

Various techniques can be introducing an —SH group into an antibody. Ina preferred one, an ε-amino group in the side chain of a lysine residuein the antibody is reacted with 2-iminothiolane to introduce a freethiol (—SH) group. The thiol group can react with a maleimide or othernucleophile acceptor group to effect conjugation:

Typically, a thiolation level of two to three thiols per antibody isachieved. For a representative procedure, see Cong et al. 2014, thedisclosure of which is incorporated herein by reference. Thus, in oneembodiment, an antibody for conjugation to a dimer of this invention hasone or more lysine residues (preferably two or three) modified byreaction with iminothiolane.

An —SH group can also be used for conjugation where the antibody hasbeen modified to introduce a maleimide group thereto, in a Michaeladdition reaction that is the “mirror image” of that described above.Antibodies can be modified to have maleimide groups with N-succinimidyl4-(maleimidomethyl)-cyclohexanecarboxylate (SMCC) or its sulfonatedvariant sulfo-SMCC, both reagents being available from Sigma-Aldrich.

An alternative conjugation technique employs copper-free “clickchemistry,” in which an azide group adds across the strained alkyne bondof a cyclooctyne to form an 1,2,3-triazole ring. See, e.g., Agard etal., J. Amer. Chem. Soc. 2004, 126, 15046; Best, Biochemistry 2009, 48,6571, the disclosures of which are incorporated herein by reference. Theazide can be located on the antibody and the cyclooctyne on the drugmoiety, or vice-versa. A preferred cyclooctyne group isdibenzocyclooctyne (DIBO). Various reagents having a DIBO group areavailable from Invitrogen/Molecular Probes, Eugene, Oregon. The reactionbelow illustrates click chemistry conjugation for the instance in whichthe DIBO group is attached to the antibody (Ab):

Yet another conjugation technique involves introducing a non-naturalamino acid into an antibody, with the non-natural amino acid providing afunctionality for conjugation with a reactive functional group in thedrug moiety. For instance, the non-natural amino acidp-acetylphenylalanine can be incorporated into an antibody or otherpolypeptide, as taught in Tian et al., WO 2008/030612 A2 (2008). Theketone group in p-acetylphenyalanine can be a conjugation site via theformation of an oxime with a hydroxylamino group on the linker-drugmoiety. Alternatively, the non-natural amino acid p-azidophenylalaninecan be incorporated into an antibody to provide an azide functionalgroup for conjugation via click chemistry, as discussed above.Non-natural amino acids can also be incorporated into an antibody orother polypeptide using cell-free methods, as taught in Goerke et al.,US 2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009,102 (2), 400-416. The foregoing disclosures are incorporated herein byreference. Thus, in one embodiment, an antibody that is used for makinga conjugate with a dimer of this invention has one or more amino acidsreplaced by a non-natural amino acid, which preferably isp-acetylphenylalanine or p-azidophenylalanine, more preferablyp-acetylphenylalanine.

Still another conjugation technique uses the enzyme transglutaminase(preferably bacterial transglutaminase or BTG), per Jeger et al., Angew.Chem. Int. Ed. 2010, 49, 9995. BTG forms an amide bond between the sidechain carboxamide of a glutamine (the amine acceptor) and analkyleneamino group (the amine donor), which can be, for example, theC-amino group of a lysine or a 5-amino-n-pentyl group. In a typicalconjugation reaction, the glutamine residue is located on the antibody,while the alkyleneamino group is located on the linker-drug moiety, asshown below:

The positioning of a glutamine residue on a polypeptide chain has alarge effect on its susceptibility to BTG mediated transamidation. Noneof the glutamine residues on an antibody are normally BTG substrates.However, if the antibody is deglycosylated—the glycosylation site beingasparagine 297 (N297)—nearby glutamine 295 (Q295) is rendered BTGsusceptible. An antibody can be deglycosylated enzymatically bytreatment with PNGase F (Peptide-N-Glycosidase F). Alternatively, anantibody can be synthesized glycoside free by introducing an N297Amutation in the constant region, to eliminate the N297 glycosylationsite. Further, it has been shown that an N297Q substitution in anantibody not only eliminates glycosylation, but also introduces a secondglutamine residue (at position 297) that too is an amine acceptor. Thus,in one embodiment, an antibody that is conjugated to a dimer of thisinvention is deglycosylated. In another embodiment, the antibody has anN297Q substitution. Those skilled in the art will appreciate thatdeglycosylation by post-synthesis modification or by introducing anN297A mutation generates two BTG-reactive glutamine residues perantibody (one per heavy chain, at position 295), while an antibody withan N297Q substitution will have four BTG-reactive glutamine residues(two per heavy chain, at positions 295 and 297).

Conjugation can also be effected using the enzyme Sortase A, as taughtin Levary et al., PLoS One 2011, 6(4), e18342; Proft, Biotechnol. Lett.2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010); and Mao et al.,WO 2005/051976 A2 (2005). The Sortase A recognition motif (typicallyLPXTG, where X is any natural amino acid) may be located on the ligand Zand the nucleophilic acceptor motif (typically GGG) may be the group R³¹in formula (III), or vice-versa.

An antibody also can be adapted for conjugation by modifying itsglycosyl group to introduce a keto group that serves as a conjugationsite by oxime formation, as taught by Zhu et al., mAbs 2014, 6, 1. Inanother glycoengineering variation, an antibody's glycosyl group can bemodified to introduce an azide group for conjugation by “clickchemistry.” See Huang et al., J. Am. Chem. Soc. 2012, 134, 12308 andWang, U.S. Pat. No. 8,900,826 B2 (2014) and U.S. Pat. No. 7,807,405 B2(2010).

Yet another conjugation technique can be generally referred to asdisulfide bridging: the disulfide bonds in an antibody are cleaved,creating a pair of thiol (—SH) groups. The antibody is then treated witha drug-linker compound that contains two thiol-reactive sites. Reactionof the thiol groups with the two sites effects a re-bridging thatre-creates, in a fashion, the original disulfide bridge, thus preservingthe antibody tertiary structure and attaching a drug-linker moiety. See,e.g., Burt et al., WO 2013/190292 A2 (2013) and Jackson et al., US2013/0224228 A1 (2013).

Dimer-Linker Compounds

Generally, an ADC of a dimer of this invention comprises a linkerattached to a functional group on the dimer, which linker is attached tothe antibody. Reflecting the diversity of conjugation techniquesavailable, the dimers of this invention can be elaborated into manydifferent dimer-linker compounds suitable for conjugation to anantibody.

Generally, there are three different modes for attachment of the linkerto a dimer of this invention, as illustrated in the figure below (whichis a simplified version of formula I with some variables not shown forsimplicity):

In type (a) and (a′) dimer-linker compounds, a functional group forattachment of the linker is located in the bridge X or Y between the twodimer halves. In type (b) dimer-linker compounds, the linker is attachedas an addition product across an imine double bond. In type (c)dimer-linker compounds, a functional group for attachment of the linkeris located at either A or B.

A preferred dimer-linker compound has a structure according to fomulaIII:

wherein

-   R⁶° is according to formula IIIa, IIIa′, or IIIa″

-   Y is (CH₂)₆₋₁₀ (preferably (CH₂)₈);-   x is 3 or 5 (preferably 3);-   each y is independently 2, 3, or 4 (preferably both are 4);-   A and B are independently according to formula Ia or Ib

-   -   wherein, in formula Ia    -   Y′ and Y″ are independently absent, CH₂, C═O, or CHR¹²; wherein        each R¹² is independently F, Cl, Br, or C₁-C₃ alkyl, with the        proviso that Y′ and Y″ are not both absent;    -   each G is independently C or N, with the proviso that no more        than two Gs are N; and    -   each R⁵, R⁶, R⁷, and R⁸ is independently H, Cl, Br, C₁₋₃ alkyl,        NO₂, CN, NH₂, O(C₁₋₃ alkyl), or (OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl)        (preferably H);        -   or where a R⁵, R⁶, R⁷, or R⁸ is attached to—i.e., is            associated with—a G that is N, such R⁵, R⁶, R⁷, or R⁸ is            absent;    -   and    -   wherein, in formula Ib,    -   the dotted lines indicate the optional presence of a C1-C2,        C2-C3, or C2-R¹⁰ double bond;    -   R⁹ is absent if a C1-C2, C2-C3, or C2-R¹⁰ double bond is present        and otherwise is H;    -   R¹⁰ is H, Cl, Br, ═CH₂, ═CH(C₁₋₅ alkyl), C₁₋₃ alkyl, NO₂, CN, or        NH₂ (preferably H);

-   A′ is

-   R⁵⁰ is H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂, O(C₁₋₃ alkyl), or    (OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl) (preferably H);-   R⁵¹ is H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, or NH₂ (preferably H);-   T is a self-immolating group;-   t is 0 or 1;-   AA^(a) and each AA^(b) are independently selected from the group    consisting of alanine, β-alanine, γ-aminobutyric acid, arginine,    asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline,    cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,    leucine, lysine, methionine, norleucine, norvaline, ornithine,    phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and    valine;-   p is 1, 2, 3, or 4;-   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 2, 3, 4, or 8);-   r is 1, 2, 3, 4, or 5 (preferably 2, 3, 4, or 5);-   s is 0 or 1; and-   R³¹ is

In a preferred dimer-linker compound according to formula III, R⁶⁰ isIIIa, corresponding to a dimer-linker of the following structure:

In another preferred dimer-linker compound according to formula III, R⁶⁰is IIIa′, corresponding to a dimer-linker of the following structure:

In yet another preferred dimer-linker compound according to formula III,R⁶⁰ is IIIa″, corresponding to a dimer-linker of the followingstructure:

R³¹ in formula III is a reactive functional group capable of reactingwith a complementary functional group on the antibody to effectconjugation, as described above.

In formula III, -AA^(a)-[AA^(b)]_(p)- represents a polypeptide whoselength is determined by the value of p (dipeptide if p is 1,tetrapeptide if p is 3, etc.). AA^(a) is at the carboxy terminus of thepolypeptide and its carboxyl group forms a peptide (amide) bond with anamine nitrogen of the dimer. Conversely, the last AA^(b) is at the aminoterminus of the polypeptide and its α-amino group forms a peptide bondwith

depending on whether s is 1 or 0, respectively. Preferred polypeptides-AA^(a)-[AA^(b)]_(p)- are Val-Cit, Val-Lys, Lys-Val-Ala, Asp-Val-Ala,Val-Ala, Lys-Val-Cit, Ala-Val-Cit, Val-Gly, Val-Gln, and Asp-Val-Cit,written in the conventional N-to-C direction, as in H₂N-Val-Cit-CO₂H).More preferably, the polypeptide is Val-Cit, Val-Lys, or Val-Ala.Preferably, a polypeptide -AAa-[AA^(b)]_(p)- is cleavable by an enzymefound inside the target (cancer) cell, for example a cathepsin andespecially cathepsin B.

As indicated by the subscript t equals 0 or 1, a self-immolating group Tis optionally present in dimer-linker compounds of formula III. Whenpresent, the self-immolating group T preferably is a p-aminobenzyloxycarbonyl (PABC) group, whose structure is shown below, with anasterisk (*) denoting the end of the PABC bonded to an amine nitrogen ofthe dimer and a wavy line

denoting the end bonded to the polypeptide -AA^(a)-[AA^(b)]_(p)-.

Preferred dimer linker compounds are selected from the group havingstructures represented by formulae IIIa-1, IIIa-3, IIIa-4, IIIa-5,IIIa-6, and IIIa-7.

Preparation of Conjugates

This general procedure is based on introduction of free thiol groupsinto an antibody by reaction of lysine ε-amino groups with2-iminothiolane, followed by reaction with a maleimide-containingdrug-linker moiety, such as described above. Initially the antibody isbuffer exchanged into 0.1 M phosphate buffer (pH 8.0) containing 50 mMNaCl and 2 mM diethylene triamine pentaacetic acid (DTPA) andconcentrated to 5-10 mg/mL. Thiolation is achieved through addition of2-iminothiolane to the antibody. The amount of 2-iminothiolane to beadded can be determined by a preliminary experiment and varies fromantibody to antibody. In the preliminary experiment, a titration ofincreasing amounts of 2-iminothiolane is added to the antibody, andfollowing incubation with the antibody for 1 h at RT (room temperature,circa 25° C.), the antibody is desalted into 50 mM HEPES, 5 mM Glycine,2 mM DTPA, pH 5.5 using a SEPHADEX™ G-25 column and the number of thiolgroups introduced determined rapidly by reaction with dithiodipyridine(DTDP). Reaction of thiol groups with DTDP results in liberation ofthiopyridine, which can be monitored spectroscopically at 324 nm.Samples at a protein concentration of 0.5-1.0 mg/mL are typically used.The absorbance at 280 nm can be used to accurately determine theconcentration of protein in the samples, and then an aliquot of eachsample (0.9 mL) is incubated with 0.1 mL DTDP (5 mM stock solution inethanol) for 10 min at RT. Blank samples of buffer alone plus DTDP arealso incubated alongside. After 10 min, absorbance at 324 nm is measuredand the number of thiol groups is quantitated using an extinctioncoefficient for thiopyridine of 19,800 M⁻¹.

Typically a thiolation level of about two to three thiol groups perantibody is desirable. For example, with some antibodies this can beachieved by adding a 15-fold molar excess of 2-iminothiolane followed byincubation at RT for 1 h. The antibody is then incubated with2-iminothiolane at the desired molar ratio and then desalted intoconjugation buffer (50 mM HEPES, 5 mM glycine, 2 mM DTPA, pH 5.5)). Thethiolated material is maintained on ice while the number of thiolsintroduced is quantitated as described above.

After verification of the number of thiols introduced, the drug(dimer)-linker moiety is added at a 2.5-fold molar excess per thiol. Theconjugation reaction is allowed to proceed in conjugation buffercontaining a final concentration of 25% propylene glycol and 5%trehalose. Commonly, the drug-linker stock solution is dissolved in 100%DMSO. The stock solution is added directly to the thiolated antibody.

The conjugation reaction mixture is incubated at RT for 2 h with gentlestirring. A 10-fold molar excess of N-ethyl maleimide (100 mM Stock inDMSO) is then added to the conjugation mixture and stirred for anadditional hour to block any unreacted thiols.

The sample is then filtered via a 0.2μ filter The material is bufferexchanged via TFF VivaFlow 50 Sartorius 30 MWCO PES membrane into 10mg/mL glycine, 20 mg/mL sorbitol, 15% acetonitrile (MeCN) pH 5.0 (5× TFFbuffer exchange volume), to remove any unreacted drug. The finalformulation is carried out by TFF into 20 mg/mL sorbitol, 10 mg/mLglycine, pH 5.0.

Those skilled in the art will understand that the above-describedconditions and methodology are exemplary and non-limiting and that otherapproaches for conjugation are known in the art and usable in thepresent invention.

A preferred conjugate of this invention has a structure represented byformula IV:

wherein

-   Ab is an antibody;-   m is 1, 2, 3, or 4;-   R⁴⁰ is

-   -   where the open valence of R⁴⁰ that is bonded to Ab is denoted by        an asterisk (*) and the open valence of R⁴⁰ that is bonded to        (CH₂)_(r) is denoted by a wavy line        ;

-   R⁶° is according to formula IIIa, IIIa′, or IIIa″

-   Y is (CH₂)₆₋₁₀ (preferably (CH₂)8);-   x is 3 or 5 (preferably 3);-   each y is independently 2, 3, or 4 (preferably both are 4);-   A and B are independently according to formula Ia or Ib

-   -   wherein, in formula Ia    -   Y′ and Y″ are independently absent, CH₂, C═O, or CHR¹²; wherein        each R′² is independently F, Cl, Br, or C₁-C₃ alkyl, with the        proviso that Y′ and Y″ are not both absent;    -   each G is independently C or N, with the proviso that no more        than two Gs are N; and    -   each R⁵, R⁶, R⁷, and R⁸ is independently H, Cl, Br, C₁₋₃ alkyl,        NO₂, CN, NH₂, O(C₁₋₃ alkyl), or (OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl)        (preferably H);        -   or where a R⁵, R⁶, R⁷, or R⁸ is attached to—i.e., is            associated with—a G that is N, such R⁵, R⁶, R⁷, or R⁸ is            absent;    -   and    -   wherein, in formula Ib,    -   the dotted lines indicate the optional presence of a C1-C2,        C2-C3, or C2-R¹⁰ double bond;    -   R⁹ is absent if a C1-C2, C2-C3, or C2-R¹⁰ double bond is present        and otherwise is H;    -   R¹⁰ is H, Cl, Br, ═CH₂, ═CH(C₁₋₅ alkyl), C₁₋₃ alkyl, NO₂, CN, or        NH₂ (preferably H);

-   A′ is

-   R⁵⁰ is H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂, O(C₁₋₃ alkyl), or    (OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl) (preferably H);-   R⁵¹ is H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂ (preferably H);-   T is a self-immolating group;-   t is 0 or 1;-   AA^(a) and each AA^(b) are independently selected from the group    consisting of alanine, β-alanine, γ-aminobutyric acid, arginine,    asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline,    cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,    leucine, lysine, methionine, norleucine, norvaline, ornithine,    phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and    valine;-   p is 1, 2, 3, or 4;-   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 2, 3, 4, or 8);

r is 1, 2, 3, 4, or 5 (preferably 2, 3, 4, or 5); and

-   s is 0 or 1.

In a preferred conjugate according to formula IV, R⁶⁰ is IIIa,corresponding to a conjugate having a structure represented by formulaIVa:

In another preferred conjugate according to formula IV, R⁶⁰ is IIIa′,corresponding to a conjugate having a structure represented by formulaIVa′:

In another preferred conjugate according to formula IV, R⁶⁰ is IIIa″,corresponding to a conjugate having a structure represented by formulaIVa″:

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the present invention, or of aconjugate thereof, formulated together with a pharmaceuticallyacceptable carrier or excipient. It may optionally contain one or moreadditional pharmaceutically active ingredients, such as an antibody oranother drug. The pharmaceutical compositions can be administered in acombination therapy with another therapeutic agent, especially anotheranti-cancer agent.

The pharmaceutical composition may comprise one or more excipients.Excipients that may be used include carriers, surface active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

Preferably, a pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound may be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” means modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Alternatively, the pharmaceuticalcomposition can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically.

Pharmaceutical compositions can be in the form of sterile aqueoussolutions or dispersions. They can also be formulated in amicroemulsion, liposome, or other ordered structure suitable to achievehigh drug concentration. The compositions can also be provided in theform of lyophilates, for reconstitution in water prior toadministration.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 0.01 percent to about ninety-nine percent of activeingredient, preferably from about 0.1 percent to about 70 percent, mostpreferably from about 1 percent to about 30 percent of active ingredientin combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide a therapeutic response. Forexample, a single bolus may be administered, several divided doses maybe administered over time, or the dose may be proportionally reduced orincreased as indicated by the exigencies of the situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. “Dosageunit form” refers to physically discrete units suited as unitary dosagesfor the subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic response, in association with the required pharmaceuticalcarrier.

The dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg, of the host body weight. For example dosages can be 0.3mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kgbody weight or 10 mg/kg body weight or within the range of 1-10 mg/kg,or alternatively 0.1 to 5 mg/kg. Exemplary treatment regimens areadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months, or onceevery three to 6 months. Preferred dosage regimens include 1 mg/kg bodyweight or 3 mg/kg body weight via intravenous administration, using oneof the following dosing schedules: (i) every four weeks for six dosages,then every three months; (ii) every three weeks; (iii) 3 mg/kg bodyweight once followed by 1 mg/kg body weight every three weeks. In somemethods, dosage is adjusted to achieve a plasma antibody concentrationof about 1-1000 μg/mL and in some methods about 25-300₁1μ/mL.

A “therapeutically effective amount” of a compound of the inventionpreferably results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.For example, for the treatment of tumor-bearing subjects, a“therapeutically effective amount” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. A therapeutically effectiveamount of a therapeutic compound can decrease tumor size, or otherwiseameliorate symptoms in a subject, which is typically a human but can beanother mammal.

The pharmaceutical composition can be a controlled or sustained releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See,e.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3)transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparati(U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S.Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which areincorporated herein by reference.

In certain embodiments, the pharmaceutical composition can be formulatedto ensure proper distribution in vivo. For example, to ensure that thetherapeutic compounds of the invention cross the blood-brain barrier,they can be formulated in liposomes, which may additionally comprisetargeting moieties to enhance selective transport to specific cells ororgans. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and5,399,331; V. V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa etal., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al.(1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. AgentsChemother. 39:180; Briscoe et al. (1995) Am. J. Physiol. 1233:134;Schreier et al. (1994 J. Biol. Chem. 269:9090; Keinanen and Laukkanen(1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods4:273.

Uses

Compounds of this invention or their conjugates can be used for treatingdiseases such as, but not limited to, hyperproliferative diseases,including: cancers of the head and neck which include tumors of thehead, neck, nasal cavity, paranasal sinuses, nasopharynx, oral cavity,oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas;cancers of the liver and biliary tree, particularly hepatocellularcarcinoma; intestinal cancers, particularly colorectal cancer; ovariancancer; small cell and non-small cell lung cancer (SCLC and NSCLC);breast cancer sarcomas, such as fibrosarcoma, malignant fibroushistiocytoma, embryonal rhabdomyosarcoma, leiomysosarcoma,neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, andalveolar soft part sarcoma; leukemias such as acute promyelocyticleukemia (APL), acute myelogenous leukemia (AML), acute lymphoblasticleukemia (ALL), and chronic myelogenous leukemia (CML); neoplasms of thecentral nervous systems, particularly brain cancer; multiple myeloma(MM), lymphomas such as Hodgkin's lymphoma, lymphoplasmacytoid lymphoma,follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, mantlecell lymphoma, B-lineage large cell lymphoma, Burkitt's lymphoma, andT-cell anaplastic large cell lymphoma. Clinically, practice of themethods and use of compositions described herein will result in areduction in the size or number of the cancerous growth and/or areduction in associated symptoms (where applicable). Pathologically,practice of the method and use of compositions described herein willproduce a pathologically relevant response, such as: inhibition ofcancer cell proliferation, reduction in the size of the cancer or tumor,prevention of further metastasis, and inhibition of tumor angiogenesis.The method of treating such diseases comprises administering atherapeutically effective amount of an inventive combination to asubject. The method may be repeated as necessary. Especially, the cancercan be renal, lung, gastric, or ovarian cancer.

Compounds of this invention or their conjugates can be administered incombination with other therapeutic agents, including antibodies,alkylating agents, angiogenesis inhibitors, antimetabolites, DNAcleavers, DNA crosslinkers, DNA intercalators, DNA minor groove binders,enediynes, heat shock protein 90 inhibitors, histone deacetylaseinhibitors, immuno-modulators, microtubule stabilizers, nucleoside(purine or pyrimidine) analogs, nuclear export inhibitors, proteasomeinhibitors, topoisomerase (I or II) inhibitors, tyrosine kinaseinhibitors, and serine/threonine kinase inhibitors. Specific therapeuticagents include adalimumab, ansamitocin P3, auristatin, bendamustine,bevacizumab, bicalutamide, bleomycin, bortezomib, busulfan, callistatinA, camptothecin, capecitabine, carboplatin, carmustine, cetuximab,cisplatin, cladribin, cytarabin, cryptophycins, dacarbazine, dasatinib,daunorubicin, docetaxel, doxorubicin, duocarmycin, dynemycin A,epothilones, etoposide, floxuridine, fludarabine, 5-fluorouracil,gefitinib, gemcitabine, ipilimumab, hydroxyurea, imatinib, infliximab,interferons, interleukins, β-lapachone, lenalidomide, irinotecan,maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate,mitomycin C, nilotinib, oxaliplatin, paclitaxel, procarbazine,suberoylanilide hydroxamic acid (SAHA), 6-thioguanidine, thiotepa,teniposide, topotecan, trastuzumab, trichostatin A, vinblastine,vincristine, and vindesine.

EXAMPLES

The practice of this invention can be further understood by reference tothe following examples, which are provided by way of illustration andnot of limitation. The following general procedures are illustrative,with thoseskilled in the art understanding that alternative butequivalent methods can be used.

Example 1 Dimer IIa-9

This example pertains to FIGS. 1A-1B and the synthesis of dimer IIa-9.

4-(Benzyloxy)-5-methoxy-2-nitrobenzoyl chloride 1 was prepared from thecorresponding methyl ester as follows: To a solution of methyl4-(benzyloxy)-5-methoxy-2-nitrobenzoate (Harve Chem, 15 g, 47.3 mmol) intetrahydrofuran (THF, 350 mL) was added a solution of aq. NaOH (56.7 mL,142 mmol, 2.5M). The reaction was stirred at 50° C. for 5 h. Thereaction was cooled to room temperature (RT) and then concentrated invacuo to remove the THF. The remaining aqueous layer was acidified withaq. HCl (6 N) to pH 2. The resulting yellow precipitate was filtered,washed with water, and dried under vacuum to give4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (14.32 g, 100% yield). LCMS(M+H)=304.08; ¹H NMR (400 MHz, METHANOL-d4) δ 7.60 (s, 1H), 7.53-7.45(m, 2H), 7.45-7.31 (m, 3H), 7.29 (s, 1H), 5.23 (s, 2H), 3.98 (s, 3H).

To a solution of the above nitrobenzoic acid (1.2 g, 3.96 mmol) in THF(30 mL) was added dropwise oxalyl chloride (0.416 mL, 4.75 mmol),followed by N,N-dimethyl-formamide (DMF, 20 uL). The resulting solutionwas stirred at RT for 35 h before it was concentrated in vacuo to giveacid chloride 1 as a yellow solid.

Acid chloride 1 was dissolved in THF (20 mL) and added dropwise to asolution of of S-methyl pyrrolidine-2-carboxylate 2 hydrochloride (0.768g, 4.75 mmol) and triethylamine (NEt₃, 1.65 mL, 11.87 mmol) in THF (10mL) at 0° C. The reaction mixture was warmed to RT and stirred at RT for1 h before quenching with aq. LiCl and concentrated to remove the THF.The resulting mixture was extracted with EtOAc (3×). The combinedorganic layers were washed with sat. aq. NaHCO₃ and then brine and driedover Na2SO₄ and concentrated in vacuo. The crude product mixture waspurified using ISCO silica gel chromatography (80 g column, gradientfrom 0% to 100% EtOAc/dichloromethane (DCM) in 15 minutes) to give ester3 (1.18 mg, 72% yield). LCMS (M+H)=415.4; 41 NMR (400 MHz, CHLOROFORM-d)δ 7.77 (s, 1H), 7.51-7.32 (m, 5H), 6.92-6.80 (m, 1H), 5.25-5.20 (m, 2H),4.80-4.73 (m, 1H), 4.03-3.93 (m, 3H), 3.82 (s, 2H), 3.56 (s, 1H),3.38-3.30 (m, 1H), 3.21 (s, 1H), 2.41-2.30 (m, 1H), 2.16-2.07 (m, 1H),2.04-1.87 (m, 2H).

A suspension of ester 3 (900 mg, 2.172 mmol) and Pd(OH)₂ (20% on carbon,90 mg) in EtOH (15 mL) was stirred under H₂ (50 psi) for 3 h. Thereaction mixture was filtered through a pad of CELITE™ and washed withEtOAc. The combined filtrates were concentrated and dissolved in MeOH(10 mL). After a drop of AcOH was added, the reaction was heated at 80°C. for 5 h. The reaction was then cooled to RT and concentrated. Theresidue was purified using ISCO silica gel chromatography (40 g column,0-100% EtOAc/Hexane gradient) to give compound 4. LCMS (M+H)=263; ¹H NMR(400 MHz, CHLOROFORM-d) δ 10.18 (s, 1H), 9.89 (br. s., 1H), 7.22 (s,1H), 6.55 (s, 1H), 4.17-3.96 (m, 1H), 3.77 (s, 3H), 3.63-3.37 (m, 2H),1.96-1.63 (m, 4H).

A suspension of compound 4 (0.8 g, 3.05 mmol) and 1,3-bromopropane 4a(0.308 g, 1.525 mmol) and K₂CO₃ (527 mg, 3.81 mmol) in DMSO (8 mL) werestirred at RT for 12 h. The reaction mixture was diluted with aq. NH₄Cland extracted with chloroform (3×). The combined organic layers werewashed with brine, dried over Na₂SO₄, and concentrated in vacuo. Thecrude product mixture was purified using ISCO silica gel chromatography(gradient from 0% to 10% MeOH/DCM) to give compound 5 (670 mg, 78%yield). LCMS (M+H)=565; ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.45 (s, 2H),6.54 (s, 2H), 4.19 (d, J=7.5 Hz, 4H), 4.03 (d, J=5.9 Hz, 2H), 3.89 (s,6H), 3.82-3.72 (m, 2H), 3.64-3.55 (m, 2H), 2.73 (br. s., 2H), 2.37-2.32(m, 2H), 2.07-1.95 (m, 6H).

To a solution of compound 5 (650 mg, 1.151 mmol) in DCM (2 mL) at −78°C. was added dropwise a solution of boron tribromide (10.04 mL, 10.4mmol, 1M in DCM). The reaction was slowly warmed to −5° C. and stirredfor 30 min. The reaction was then quenched with aq. potassium phosphatedibasic buffer (5 mL), and then concentrated to remove DCM. Theremaining slurry was filtered to give a grey solid, which was purifiedusing ISCO silica gel chromatography (120 g column, gradient from 0% to10% MeOH/DCM) to give compound 6 (202 mg, 33% yield). LCMS (M+H)=537; ¹HNMR (400 MHz, DMSO-d₆) δ 10.10 (br. s., 2H), 9.44-9.01 (m, 2H), 7.17 (s,2H), 6.66 (s, 2H), 4.13 (br. s., 5H), 4.03 (d, J=6.4 Hz, 2H), 3.51 (br.s., 6H), 2.46 (br. s., 1H), 2.32-2.16 (m, 2H), 2.06-1.66 (m, 7H).

A suspension of compound 6 (86mg, 0.160 mmol), 6-bromohex-1-ene 6a (52.3mg, 0.321 mmol), and K₂CO₃ (66.5 mg, 0.481 mmol) in DMF (1.5 mL) wasstirred at RT for 15 h. The reaction mixture was filtered and purifiedusing ISCO silica gel chromatography (40 g column, gradient from 0% to10% MeOH/DCM) to give compound 7 (55 mg, 49% yield). LCMS (M+H)=701.7.

A vial was charged with compound 7 (52 mg, 0.074 mmol). A solution ofGrubbs-II catalyst (6.30 mg, 7.42 μmol) in DCE (10 mL) was then added.The resulting solution was degassed and heated to 75° C. for 1 h. Thereaction mixture was then concentrated and purified using ISCO silicagel chromatography (12 g column, gradient 0% to 10% MeOH/DCM) to givethe 21-membered macrocycle 8 (44 mg, 88%, along with ˜10% 20-memberedmacrocycle by-product 9). LCMS (M+H)=673.7.

To a solution of the mixture ofcompounds 8 and 9 (18mg, 0.027 mmol) inDMF (1 mL) at 0° C. was added NaH (4.01 mg, 0.067 mmol). The resultingsuspension was stirred at 0° C. for 30 min before2-(chloromethoxy)ethyl)trimethylsilane (SEM-Cl, 0.014 mL, 0.080 mmol)was added. The reaction was then stirred at 0° C. for 1 h beforequenching with brine. The mixture was extracted with EtOAc (3×). Thecombined organic layers were dried over Na₂SO₄, concentrated, andpurified using ISCO silica gel chromatography (12 g column, gradientfrom 0% to 100% MeOH/DCM) to give compound 10. LCMS (M+H)=933; ¹H NMR(400 MHz, CHLOROFORM-d) δ 7.37 (s, 2H), 7.22 (s, 2H), 5.48 (m, 2H), 5.43(t, J=3.5 Hz, 2H), 4.70 (m, 2H), 4.29-4.21 (m, 4H), 4.14-4.03 (m, 6H),3.84-3.65 (m, 7H), 3.62-3.50 (m, 2H), 2.79-2.67 (m, 2H), 2.35 (m, 2H),2.12-1.96 (m, 10H), 1.80 (m, 4H), 1.58-1.47 (m, 2H), 0.98 (m, 4H),0.04-0.02 (s, 18H).

To a solution of compound 10 in THF/EtOH (1:1, 1 mL) at 0° C. was addeda solution of lithium borohydride (0.268 mL, 0.535 mmol, 2M in THF). Theresulting solution was stirred at 0° C. for 1 h before it was warmed toRT and stirred for 5 min. The reaction was then quenched with brine andextracted with CHCl₃ (3×). The combined organic layers were dried overNa₂SO₄ and concentrated. The residue was then taken up in CHCl₃/EtOH(1:1, 2 mL). Silica gel (0.9 g) was added, followed by water (1 mL). Theresulting suspension was stirred at RT for 48 h and then filtered,washing with 10% MeOH/CHCl₃. The filtrate was concentrated and purifiedusing reverse phase HPLC (Column: Phenomenex Luna C18 20×100 mm; MobilePhase A: 10:90 MeCN:water with 0.1% trifluoroacetic acid (TFA); MobilePhase B: 90:10 MeCN:water with 0.1% TFA acid; Gradient: 0-80% B over 15minutes; Flow: 20 mL/min; Detection: UV at 220 nm) to giveThe fractionscontaining desired fractions were combined and neutralized with aq.NaHCO3, then extracted with chloroform, and dried and concentrated togive dimer IIa-9. (7.1 mg, 39% over two steps). LCMS (M+H)=641.3; ¹H NMR(400 MHz, CHLOROFORM-d) δ 7.68 (d, J=4.4 Hz, 2H), 7.53 (s, 2H), 6.85 (s,2H), 5.46 (m, 2H), 4.36-4.23 (m, 4H), 4.20-4.12 (m, 2H), 4.11-4.03 (m,2H), 3.87-3.82 (m, 2H), 3.75 (dt, J=7.5, 4.0 Hz, 2H), 3.60 (dt, J=11.8,7.8 Hz, 2H), 2.41-2.30 (m, 6H), 2.14-2.01 (m, 8H), 1.88-1.77 (m, 4H),1.67-1.58 (m, 4H).

Example 2 Dimers IIa-3 and IIa-4

This example pertains to FIG. 2 and the synthesis of dimers IIa-3 andIIa-4.

A suspension of a mixture of compounds 8 and 9 (24 mg, 0.036 mmol) and10% Pd/C (6 mg) in MeOH (3 mL) was stirred under a balloon of H₂ for 3h.The reaction mixture was purged with N₂ and filtered through a pad ofCELITE™, washing with EtOAc. The combined filtrates were concentrated togive 21-membered macrocycle 11 (24 mg, 100% yield, alon with ˜10%macrocycle 12). LCMS (M+H)=675.4.

To a solution of the mixture of macrocycle 11 and 12 (24mg, 0.036 mmol)in DMF (1 mL) at 0° C. was added NaH (5.33 mg, 0.089 mmol). Theresulting suspension was stirred at 0° C. for 30 min before SEM-Cl(0.019 mL, 0.107 mmol) was added. The reaction was stirred at 0° C. for1 h before it was quenched with brine. The mixture was extracted withEtOAc (3×). The combined organic layers were dried over Na₂SO₄,concentrated, and purified using ISCO silica gel chromatography (12 gcolumn, gradient from 0% to 100% MeOH/DCM) to give a mixture ofSEM-macrocycles (21-membered macrocycle: LCMS (M+H)=935. 20-memberedmacrocycle: LCMS (M+H)=921).

To a solution of the mixture of the above SEM-macrocycles in THF/EtOH(1:1, 1 mL) at 0° C. was added a solution of LiBH₄ (0.356 mL, 0.71 mmol,2M in THF). The resulting solution was stirred at 0° C. for 1 h beforewarming to RT and stirring for 15 min. The reaction was quenched withbrine and extracted with CHCl₃ (3×). The combined organic layers weredried over Na₂SO₄ and concentrated. The residue was taken up inCHCl₃/EtOH (1:1, 2 mL). Silica gel (0.9 g) was added, followed by water(1 mL). The resulting suspension was stirred at RT for 48 h andfiltered, washing with 10% MeOH/CHCl₃. The filtrate was concentrated andpurified using reverse phase HPLC to give dimers IIa-4 and IIa-3(Column: Phenomenex Luna C18 20×100 mm; Mobile Phase A: 10:90 MeCN:waterwith 0.1% trifluoroacetic acid (TFA); Mobile Phase B: 90:10 MeCN:waterwith 0.1% TFA acid; Gradient: 0-80% B over 15 minutes; Flow: 20 mL/min;Detection: UV at 220 nm).

Dimer IIa-4: (7.5 mg, 31% yield); LCMS (M+H)=643.4; ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.68 (d, J=4.4 Hz, 2H), 7.53 (s, 2H), 6.84 (s, 2H),4.33-4.16 (m, 6H), 4.13-4.06 (m, 2H), 3.88-3.80 (m, 2H), 3.79-3.71 (m,2H), 3.64-3.57 (m, 2H), 2.44-2.30 (m, 6H), 2.15-2.04 (m, 4H), 1.85-1.77(m, 4H), 1.61-1.52 (m, 4H), 1.44-1.38 (m, 8H).

Dimer IIa-3: (1 mg, 4% yield); LCMS (M+H)=629.4; ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.68 (d, J=4.4 Hz, 2H), 7.55 (s, 2H), 6.86 (s, 2H),4.37-4.24 (m, 4H), 4.21-4.14 (m, 2H), 4.12-4.06 (m, 2H), 3.87-3.80 (m,2H), 3.79-3.70 (m, 4H), 3.63-3.56 (m, 2H), 2.42-2.31 (m, 6H), 2.14-2.06(m, 4H), 1.83-1.78 (m, 4H), 1.61-1.52 (m, 4H), 1.43-1.33 (m, 6H).

Example 3 Dimer IIa-1

This example pertains to FIG. 3 and the synthesis of dimer IIa-1.

To a suspension of compound 6 (29mg, 0.054 mmol) and K₂CO₃ (7.47 mg,0.054 mmol) in DMF (1.5 mL) was added 1,7-dibromoheptane (14.64 mg,0.057 mmol). The mixture was heated at 50° C. for 2h. The reaction wascooled to RT. The reaction mixture was filtered and purified using ISCOsilica gel chromatography (12 g column, gradient from 0 to 10% MeOH/DCM)to give macrocycle 13. LCMS (M+H)=633.5.

To a solution of macrocycle 13 in DMF (0.8 mL) at 0° C. was added NaH(4.32 mg, 0.108 mmol). The resulting suspension was stirred at 0° C. for30 min before SEM-Cl (0.019 mL, 0.11 mmol) was added. The reaction wasstirred at 0° C. for 1 h before it was quenched with brine. The mixturewas extracted with EtOAc (3×). The combined organic layers were driedover Na₂SO₄, concentrated, and purified using ISCO silica gelchromatography (12 g column, gradient from 0% to 100% MeOH/DCM) to giveSEM-macrocycle 14 (9 mg, 10.08 μmol, 18.6% yield over two steps). LCMS(M+H)=893.4.

To a solution of SEM-macrocycle 14 (9 mg, 10.08 μmol) in THF/EtOH (1:1,1 mL) at 0° C. was added a solution of LiBH₄ (101 μL, 0.202 mmol, 2M inTHF). The resulting solution was stirred at 0° C. for 1 h before warmingto RT and stirring for 15 min. The reaction was quenched with brine andextracted with CHCl₃ (3×). The combined organic layers were dried overNa₂SO₄ and concentrated. The residue was taken up in CHCl₃/EtOH (1:1, 2mL). Silica gel (0.7 g) was added, followed by water (0.6 mL). Theresulting suspension was stirred at RT for 24 h and filtered andwashedwith 10% MeOH/CHCl₃. The filtrate was concentrated and purifiedusing reverse phase HPLC (Column: Phenomenex Luna C18 20×100mm; MobilePhase A: 10:90 MeCN:H₂O with 0.1% trifluoroacetic acid (TFA); MobilePhase B: 90:10 MeCN:water with 0.1% TFA acid; Gradient: 0-70% B over 15min; Flow: 20 mL/min; Detection: UV at 220 nm) to give dimer IIa-1 (1.8mg, 2.70 μmol, 26.8% yield). LCMS (M+H)=601.2; ¹H NMR (400 MHz,CHLOROFORM-d) δ 7.68 (d, J=4.4 Hz, 2H), 7.54 (s, 2H), 6.90 (s, 2H),4.35-4.25 (m, 4H), 4.18 (dt, J=9.5, 5.7 Hz, 2H), 4.15-4.05 (m, 2H),3.86-3.78 (m, 2H), 3.77-3.72 (m, 2H), 3.65-3.54 (m, 2H), 2.42-2.28 (m,6H), 2.14-2.03 (m, 4H), 1.91-1.80 (m, 4H), 1.70-1.61 (m, 4H), 1.49-1.41(m, 2H).

Example 4 Dimer IIb-5

This example pertains to FIGS. 4A-4B and the synthesis of dimer IIb-5.

A suspension of methyl 4-hydroxy-3-methoxybenzoate 14 (18 g, 99 mmol),K₂CO₃ (20.48 g, 148 mmol) and 1,3-dibromopropane 15 (5.04 ml, 49.4 mmol)in DMSO (300 mL) was stirred at RT for 16 hours. To the reaction mixturewas added water, and the resulting solution stirred at RT for 20 min.The resulting precipitate was filtered, washed with water and driedunder vacuum. The resulting white solid was triturated with EtOAc/DCMand filtered to give compound 16 (10.45 g, 52.4%) and a dark brownfiltrate. The filtrate was purified by ISCO (0-50% of EtOAC/DCM in 15minutes, 120 g column) to provide additional compound 16 (3.55 g,17.7%). LCMS (M+H)=405; ¹H NMR (400 MHz, DMSO-d₆) δ 7.58 (dd, J=8.4, 2.0Hz, 2H), 7.46 (d, J=2.0 Hz, 2H), 7.12 (d, J=8.6 Hz, 2H), 4.22 (t, J=6.2Hz, 4H), 3.83 (s, 6H), 3.81 (s, 6H), 2.24 (t, J=6.2 Hz, 2H);.

To a solution of tin (IV) chloride (19.91 mL, 19.91 mmol, 1M in DCM) at0° C. was added dropwise concentrated nitric acid (1.375 mL, 27.7 mmol).The resulting mixture was added dropwise to a solution of compound 16(3.5 g, 8.65 mmol) in DCM (15 mL) at −25° C. The reaction was stirred at−25° C. for 30 min befoire it was quenched with water (100 mL). Theorganic layer was separated. The aq. layer was extracted with EtOAc(2×). The combined organic layers were concentrated to give a crudeproduct, which was recrystalized from hot DCM/Hexane to give compound 17(3.5 g, 82% yield) as off white crystals. LCMS (M+H)=495; ¹H NMR (400MHz, DMSO-d₆) δ 7.68 (s, 2H), 7.32 (s, 2H), 4.29 (t, J=6.2 Hz, 4H), 3.91(s, 6H), 3.83 (s, 6H), 2.35-2.13 (m, 2H).

A flask was charged with compound 17 (3.1 g, 6.27 mmol) and aq. NaOH(25.08 mL, 62.7 mmol, 2.5 M). The reaction mixture was heated at 100° C.for 16 h. The heterogenous mixture became reddish solution at the end ofthe reaction. The reaction mixture was cooled to RT and acidified withaq. HCl to pH₂. The resulting precipitate was filtered, washed withwater and dried to give compound 18 (2.65 g, 6.05 mmol, 96% yield). ¹HNMR (400 MHz, DMSO-d₆) δ 13.41 (br. s., 2H), 10.62 (br. s., 2H), 7.59(s, 2H), 7.10 (s, 2H), 4.31 (t, J=6.2 Hz, 4H), 2.25 (quin, J=6.1 Hz,2H).

To a solution of compound 18 (2.9 g, 6.62 mmol) in THF (5 mL) at RT wasadded oxalyl chloride (1.390 mL, 15.88 mmol), followed by 2 drops ofDMF. The reaction was stirred at RT for 2 h before it was concentratedand dissolved in MeOH (20 mL). The resulting solution was stirred at RTfor 30 min and concentrated. The crude product was triturated withwater, filtered, and dried to give compound 19 (3 g, 6.43 mmol, 97%yield) LCMS (M+H)=467; ¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (br. s., 2H),7.65 (s, 2H), 7.10 (s, 2H), 4.33 (t, J=5.9 Hz, 4H), 3.80 (s, 6H),2.39-2.15 (m, 2H).

To a suspension of compound 19 (1g, 2.144 mmol) and K₂CO₃ (0.889 g, 6.43mmol) in DMF (1 mL) was added 1,4-dibromobutane 19a (3.70 g, 17.15mmol). The reaction mixture was heated to 80° C. for 2 h before it wascooled to RT, diluted with water, and extracted with EtOAc (3×). Thecombined organic layers were concentrated and purified using ISCO silicagel chromatography (40 g column, gradient from 0% to 50% EtOAC/Hexane)to give compound 20 (0.95 g, 60.2% yield). LCMS (M+H)=521; ¹H NMR (400MHz, CHLOROFORM-d) δ 7.47 (s, 2H), 7.04 (s, 2H), 4.31 (s, 5H), 4.11 (s,4H), 3.89 (s, 6H), 3.51 (t, J=6.3 Hz, 4H), 2.42 (s, 2H), 2.14-1.92 (m,8H).

A suspension of compound 20 (0.95 g, 1.290 mmol),2-nitrobenzenesulfonamide 21a (0.261 g, 1.290 mmol) and K₂CO₃ (0.535 g,3.87 mmol) in DMF (20 mL) was heated at 80° C. for 2 h. The reaction wasdiluted with water and extracted with EtOAc (3×). The combined organiclayers were concentrated and purified using ISCO silica gelchromatography (80 g column, gradient from 0% to 80% EtOAC/Hexane) togive macrocycle 21 (330 mg, 32.9% yield). LCMS (M+H)=777.5; ¹H NMR (400MHz, CHLOROFORM-d) δ 7.97 (dd, J=7.6, 1.7 Hz, 1H), 7.74-7.64 (m, 2H),7.63-7.56 (m, 1H), 7.51 (s, 2H), 7.07 (s, 2H), 4.36-4.28 (m, 4H),4.17-4.07 (m, 4H), 3.90 (s, 6H), 3.36 (br. s., 4H), 2.31 (t, J=6.1 Hz,2H), 1.86-1.80 (m., 8H)

To a solution of macrocycle 21 (320mg, 0.412 mmol) in DMF (3mL) wasadded 2-mercaptoethanol (322 mg, 4.12 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 310 μL, 2.060 mmol). Themixture was stirred at RT for 2 h, diluted with DCM, washed with waterand then brine, dried over sodium sulfate, and concentrated. The residuewas taken up in DCM (4 mL) and cooled to 0° C. Triethylamine (115 μl,0.824 mmol) was then added, followed by chloro allylformate (Alloc-Cl,99 mg, 0.824 mmol). The mixture was stirred at 0° C. for 30 min beforeit was quenched with water, extracted with DCM, dried, and concentrated.The crude product was purified using ISCO silica gel chromatography (40g column, gradient from 0% to 70% EtOAc/Hexane) to give macrocycle 22(140 mg, 0.207 mmol, 50.3% yield). LCMS (M+H)=676.2

To a suspension of macrocycle 22 in MeOH (1 mL) and THF (6 mL) was addedaq. NaOH (1M, 1 mL). The resulting mixture was stirred at RT for 12 h.The reaction mixture was concentrated to remove THF and MeOH. Theresidue was acidified with aq. HCL (1N) and extracted with EtOAc (3×).The combined organic layers were washed with brine, dried, andconcentrated to give acid 23 (135 mg, 0.208 mmol, 97% yield). LCMS(M+H)=485.

To a solution of acid 23 (135 mg, 0.208 mmol) in THF (2 mL) was addedoxalyl chloride (45.6 μl, 0.521 mmol), followed by DMF (5 uL). Thereaction mixture was stirred at RT for 2 h and concentrated. The residuewas dissolved in THF (10 mL) and cooled to 0° C. A solution of(S)-benzyl 1,2,3,4-tetrahydroisoquinoline-3-carboxylatep-toluenesulfonic acid salt 23a (Accela, 275 mg, 0.625 mmol) and NEt₃(0.29 mL, 2.09 mmol) in THF (5 mL) was added dropwise. The reactionmixture was slowly warmed to RT and stirred for 15 min before quenchingwith water. The resulting mixture was extracted with EtOAc (3×). Thecombined organic layers were dried, concentrated, and purified usingISCO silica gel chromatography (12 g column, gradient from 0% to 100%EtOAc/hexane) to give compound 24 (190 mg, 0.166 mmol, 80% yield). LCMS(M+H)=1146.8 .

A suspension of compound 24 (190mg, 0.166 mmol), zinc powder (108 mg,1.658 mmol), and NH₄Cl (133 mg, 2.486 mmol) in MeOH (4 mL) was heated to50° C. and stirred for 8 h. The reaction mixture was cooled to RT,diluted with MeOH, and filtered through a pad of CELITE™, washing withMeOH followed by EtOAc. The combined filtrates were concentrated andpurified using ISCO silica gel chromatography (24 g column, gradientfrom 0% to10% MeOH/DCM,) to give compound 25 (125 mg, 0.144 mmol, 87%yield). LCMS (M+H)=870.1; ¹H NMR (400 MHz Hz, CHLOROFORM-d) δ 7.40-7.36(m, 3H), 7.35-7.21 (m, 9H), 6.52 (br. s., 2H), 6.01-5.76 (m, 1H), 5.25(dd, J=17.2, 1.5 Hz, 1H), 5.16 (dd, J=10.5, 1.4 Hz, 1H), 5.06 (d, J=15.2Hz, 2H), 4.55 (d, J=5.5 Hz, 2H), 4.45 (d, J=15.4 Hz, 2H), 4.24-4.05 (m,4H), 4.00 (br. s., 2H), 3.49 (dd, J=15.4, 7.0 Hz, 2H), 3.31 (br. s.,4H), 3.01 (dd, J=15.4, 6.4 Hz, 2H), 2.19 (d, J=5.5 Hz, 2H), 1.84-1.76(m., 8H).

To a solution of compound 25 (120mg, 0.138 mmol) in DMF (5 mL) at 0° C.was added NaH (9.93 mg, 0.414 mmol). The resulting suspension wasstirred for 30 min before SEM-Cl (0.073 mL, 0.414 mmol) was added. Thereaction mixture was then slowly warmed to RT and stirred for 2 h beforequenching with brine. The resulting mixture was extracted with EtOAC(3×). The combined organic layers were dried, concentrated and purifiedusing ISCO silica gel chromatography (24 g column, gradient from 0% to100% EtOAc/hexane) to give an intermediate SEM-macrocycle (82 mg, 0.073mmol, 52.6% yield).

The preceding intermediate SEM macrocycle (82 mg, 0.073 mmol) wasdissolved in DCM (5 mL). The solution was purged with N2 beforePd(Ph₃P)₄ (7.97 mg, 6.90 μmol) and morpholine (0.060 mL, 0.690 mmol)were added sequentially. The reaction mixture was stirred at RTovernight before it was concentrated and purified using ISCO silica gelchromatography (12 g column, gradient from 0% to 20% MeOH/DCM) to givemacrocycle 26 (56 mg, 0.054 mmol, 38.8% yield). LCMS (M+H)=1046.3; ¹HNMR (400 MHz, CHLOROFORM-d) δ 7.40-7.27 (m, 12H), 5.51 (d, J=10.1 Hz,2H), 5.11 (d, J=15.4 Hz, 2H), 4.66 (d, J=10.1 Hz, 2H), 4.45 (d, J=15.4Hz, 2H), 4.35-4.24 (m, 6H), 4.21-4.14 (m, 2H), 4.08-3.99 (m, 2H), 3.79(d, J=6.8 Hz, 2H), 3.73-3.62 (m, 2H), 3.54 (s, 2H), 3.32-3.13 (m, 4H),3.04 (s, 2H), 2.30 (br. s., 2H), 2.18 (d, J=6.2 Hz, 4H), 1.95 (br. s.,4H), 1.65 (br. s., 4H), 0.97 (dd, J=4.2, 3.1 Hz, 4H), 0.03 (s, 18H).

To a solution of macrocycle 26 (46 mg, 44 μmol) in THF (1 mL) at −78° C.was added a solution of lithium triethylborohydride (SUPER-HYDRIDE®,0.13 mL, 0.123 mmol, 1M in THF). The resulting solution was stirred at−78° C. for 2 h before quenching with brine and extracted with CHCl₃(3×). The combined organic layers were dried over Na₂SO₄ andconcentrated. The residue was then taken up in CHCl₃/EtOH (1:1, 2 mL).Silica gel (0.8 g) was added, followed by water (0.6 mL). The resultingsuspension was stirred at RT for 24 h and then filtered, washing with10% MeOH/CHCl₃. The filtrate was concentrated and purified using reversephase HPLC (Column: Phenomenex Luna C18 20×100mm; Mobile Phase A: 10:90MeCN:water with 0.1% TFA; Mobile Phase B: 90:10 MeCN:water with 0.1%TFA; Gradient: 10-70% B over 15 min; Flow: 20 mL/min; Detection: UV at220 nm). to give dimer IIb-5 (20 mg, 24 μmol, 54.3% yield). LCMS(M+H)=754.2; ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.54 (s, 2H), 7.49 (d,J=5.3 Hz, 2H), 7.42-7.30 (m, 8H), 6.84 (s, 2H), 5.03 (d, J=15.6 Hz, 2H),4.56 (d, J=15.4 Hz, 2H), 4.37-4.20 (m, 4H), 4.15-4.07 (m, 2H), 4.02-3.94(m, 2H), 3.36-3.25 (m, 2H), 3.23-3.13 (m, 2H), 3.05-2.95 (m, 6H),2.42-2.30 (m, 2H), 1.96 (br. s., 8H).

Example 5 Intermediate Compound 48

This example pertains to FIG. 5 and the synthesis of intermediatecompound 48.

To a RT solution of compound 19 (10.36 g, 22.21 mmol) in DMF (178 mL)was added K₂CO₃ (12.28 g, 89 mmol). The mixture was heated to 100° C.,then a solution of 1,8-diiodooctane 19b (5.30 mL, 26.7 mmol) in DMF(44.4 mL) was added dropwise over 1.5 h. The reaction mixture wasstirred at 100° C. for 3 h more, cooled to RT, and slowly added to astirred flask of H₂O (2000 mL). The resulting precipitate was collectedby vacuum filtration (washed with H₂O). The crude material was purifiedby flash chromatography (220 g silica gel; linear gradient 0-10%EtOAc-DCM) to provide macrocycle 43 (5.994 g, 47%) as a white solid.LC-MS m/z 594 [M+18]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.53 (s, 2H), 7.08 (s,2H), 4.33 (t, J=6.1 Hz, 4H), 4.15 (t, J=5.3 Hz, 4H), 3.91 (s, 6H), 2.36(quin, J=6.0 Hz, 2H), 1.86-1.78 (m, 4H), 1.61-1.52 (m, 4H), 1.49-1.42(m, 4H).

To a RT suspension of macrocycle 43 (5.994 g, 10.40 mmol) in THF (78 mL)was added MeOH (26.0 mL) followed by 1 M aq. NaOH (104 mL, 104 mmol).The yellow suspension was stirred at 50° C. for 4 h, gradually becominga clear yellow solution. The reaction mixture was cooled to RT,partially concentrated, and acidified with 1 M aq. HCl. The resultingsolids were collected by vacuum filtration (washed with H₂O) to providemacrocycle 44 (5.41 g, 95%) as a yellow solid. LC-MS m/z 566 [M+18]⁺; ¹HNMR (400 MHz, DMSO-d₆) δ 13.58 (br s, 2H), 7.64 (s, 2H), 7.30 (s, 2H),4.27 (t, J=6.2 Hz, 4H), 4.16 (t, J=5.1 Hz, 4H), 2.20 (quin, J=6.1 Hz,2H), 1.74-1.65 (m, 4H), 1.53-1.44 (m, 4H), 1.42-1.32 (m, 4H).

To a RT solution of macrocycle 44 (5.144 g, 9.38 mmol) in THF (94 mL)was added oxalyl chloride (2.140 mL, 22.51 mmol) followed by DMF (7.29μL, 0.094 mmol). The reaction mixture was stirred at RT for 1 h thenconcentrated in vacuo. The residue was taken up in THF (94 mL) andcooled to 0° C. NEt₃ (7.84 mL, 56.3 mmol) and compound 44a (5.84 g,22.51 mmol) were added. The cooling bath was removed and the reactionmixture was stirred at RT for 3 h. The reaction was quenched by theaddition of a mixture of sat. aq. NH₄Cl (250 mL) and H₂O (250 mL) andextracted with EtOAc (2×250 mL). The combined organic layers were washedwith sat. aq. NaCl (250 mL), dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude material was purified by flashchromatography (120 g silica gel; linear gradient 0-100% EtOAc-hexanes)to provide compound 45 (9.274 g, 96%) as a yellow foam. LC-MS m/z 1031[M+H]⁺.

To a 0° C. solution of compound 45 (9.274 g, 8.99 mmol) in MeOH (56.2mL) and THF (56.2 mL) was added NH₄Cl (9.62 g, 180 mmol) and zinc dust(11.76 g, 180 mmol). The resulting suspension was stirred at 50° C. for22 h. The reaction was cooled to RT and filtered through CELITE™ (washedwith 300 mL EtOAc). The filtrate was concentrated in vacuo. The crudematerial was taken up in DCM (400 mL), washed with H₂O (400 mL), driedover Na₂SO₄, filtered, and concentrated in vacuo to provide compound 46(8.2 g, quantitative yield) as a yellow foam. LC-MS m/z 907 [M+H]⁺; ¹HNMR (400 MHz, DMSO-d₆) δ 10.25 (s, 2H), 7.27 (s, 2H), 6.73 (s, 2H), 4.46(quin, J=5.2 Hz, 2H), 4.24-4.12 (m, 6H), 4.06-3.96 (m, 4H), 3.60-3.53(m, 2H), 3.50-3.43 (m, 2H), 2.69-2.56 (m, 2H), 2.26-2.16 (m, 2H),1.98-1.89 (m, 2H), 1.72-1.62 (m, 4H), 1.54-1.43 (m, 4H), 1.41-1.32 (m,4H), 0.87-0.83 (m, 18H), 0.08 (s, 12H).

To a 0° C. solution of compound 46 (8.16 g, 8.99 mmol) in DMF (90 mL)was added NaH (1.798 g, 60% w/w in mineral oil, 45.0 mmol). The reactionmixture was stirred at 0° C. for 30 min and SEM-Cl (6.38 mL, 36.0 mmol)was added dropwise. The reaction mixture was stirred at 0° C. for 30min. Reaction was quenched by the dropwise addition of saturated aq.NH₄Cl, followed by warming to RT, dilution with EtOAc (400 mL), washingwith H₂O (2×400 mL) and sat. aq. NaCl (200 mL), drying over Na₂SO₄,filtering, and concentrating in vacuo. The crude material was purifiedby flash chromatography (220 g silica gel; linear gradient 0-100%EtOAc-hexanes) to provide compound 47 (7.980 g, 76%) as a white foam.LC-MS m/z 1168[M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (s, 2H), 7.24 (s,2H), 5.48 (d, J=10.0 Hz, 2H), 4.69 (d, J=9.9 Hz, 2H), 4.58 (quin, J=5.7Hz, 2H), 4.31-4.21 (m, 6H), 4.19-4.06 (m, 4H), 3.82-3.63 (m, 6H), 3.56(dd, J=11.9, 5.6 Hz, 2H), 2.90-2.81 (m, 2H), 2.33 (quin, J=6.0 Hz, 2H),2.07-1.98 (m, 2H), 1.84-1.75 (m, 4H), 1.62-1.54 (m, 4H), 1.50-1.40 (m,4H), 1.01-0.95 (m, 4H), 0.88 (s, 18H), 0.10 (s, 12H), 0.04 (s, 18H).

To a RT solution of compound 47 (7.979 g, 6.83 mmol) in THF (68.3 mL)was added tetrabutylammonium fluoride (TBAF, 17.08 mL, 1 M solution inTHF, 17.08 mmol). The clear yellow solution was stirred at RT for 15 h.The reaction mixture was diluted with DCM (400 mL), washed with H₂O (400mL) and sat. aq. NaCl (400 mL), dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude material was purified by flashchromatography (120 g silica gel with 25 g prepacked load cartridge;linear gradient 0-10% MeOH—CH₂Cl₂) to provide compound 48 (5.360 g) as awhite foam. The combined aqueous layers from the workup were extractedwith DCM (250 mL). The organic layer was dried over Na₂SO₄, filtered,and concentrated in vacuo. This material was purified by flashchromatography (3 ×) (80 g silica gel with 25 g prepacked loadcartridge; linear gradient 0-10% MeOH—CH₂Cl₂) to provide an additional0.617 g compound 48. The two isolates were combined to provide compound48 (5.997 g, 93%) as a white foam. LC-MS m/z 939 [M+H]⁺; ¹H NMR (400MHz, CDCl₃) δ 7.37 (s, 2H), 7.25 (s, 2H), 5.48 (d, J=10.0 Hz, 2H), 4.70(d, J=10.0 Hz, 2H), 4.69-4.64 (m, 2H), 4.33-4.25 (m, 6H), 4.16-4.03 (m,4H), 3.85 (dd, J=12.7, 2.1 Hz, 2H), 3.78 (td, J=9.6, 7.1 Hz, 2H),3.72-3.63 (m, 4H), 2.97 (dt, J=13.6, 5.5 Hz, 2H), 2.32 (quin, J=6.1 Hz,2H), 2.16-2.07 (m, 2H), 1.95-1.87 (m, 2H), 1.84-1.74 (m, 4H), 1.63-1.53(m, 4H), 1.48-1.42 (m, 4H), 0.99 (ddd, J=9.5, 6.9, 2.3 Hz, 4H),0.08-−0.01 (m, 18H).

Example 6 Dimers IIc-7 and IIc-8

This example pertains to FIG. 6 and the synthesis of dimers IIc-7 andIIc-8.

To a 0° C. solution of compound 48 (5.997 g, 6.38 mmol) in DCM (31.9 mL)and DMSO (31.9 mL) was added NEt₃ (8.90 mL, 63.8 mmol) followed bySO₃-pyridine complex (4.06 g, 25.5 mmol). The reaction was allowed towarm to RT as it was stirred for 16 h. The reaction was diluted with DCM(400 mL), washed with sat. aq. NH₄Cl (400 mL), H₂O (2×400 mL), and sat.aq. NaHCO₃ (400 mL), dried over Na₂SO₄, filtered, and concentrated invacuo. The crude material was purified by flash chromatography (120 gsilica gel with 25 g prepacked load cartridge; linear gradient 0-100%EtOAc-CH₂Cl₂) to provide compound 54 (4.822 g, 81%) as a white foam.LC-MS m/z 935 [M+H]⁻⁰ ; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (s, 2H), 7.28 (s,2H), 5.52 (d, J=10.1 Hz, 2H), 4.76 (d, J=10.0 Hz, 2H), 4.65 (dd, J=9.8,3.0 Hz, 2H), 4.36-4.28 (m, 4H), 4.28-4.20 (m, 2H), 4.18-4.06 (m, 4H),3.94-3.86 (m, 2H), 3.78 (td, J=9.8, 6.7 Hz, 2H), 3.69 (td, J=9.8, 6.6Hz, 2H), 3.58 (dd, J=19.1, 2.9 Hz, 2H), 2.85-2.73 (m, 2H), 2.39-2.31 (m,2H), 1.86-1.77 (m, 4H), 1.64-1.53 (m, 4H), 1.50-1.42 (m, 4H), 0.99 (ddd,6.6, 4.7 Hz, 4H), 0.04 (s, 18H).

To a −78° C. solution of compound 54 (4.822 g, 5.16 mmol) in DCM (129mL) was added 2,6-lutidine (3.72 mL, 32.0 mmol) andtrifluoromethanesulfonic anhydride (Tf₂O, 30.9 mL, 1 M solution in DCM,30.9 mmol) dropwise over 30 min. The bright yellow solution was allowedto warm to −20° C. over 1.5 h, then it was stirred at −20° C. for anadditional 1 h. The reaction was diluted with sat. aq. NaHCO₃ (400 mL)and extracted with DCM (2×200 mL). The combined organic layers werewashed with H₂O (200 mL), dried over Na₂SO₄, filtered, and concentratedin vacuo. The crude material was purified by flash chromatography (2×)(120 g silica gel with 25 g prepacked load cartridge; linear gradient0-100% EtOAc-hexanes) to provide compound 55 (4.018 g, 65%) as an orangefoam. ¹H NMR (400 MHz, DMSO-d₆) δ 7.38 (t, J=2.0 Hz, 2H), 7.28 (s, 2H),7.25 (s, 2H), 5.33-5.26 (m, 2H), 5.21 (d, J=10.5 Hz, 2H), 4.91 (dd,J=10.8, 3.5 Hz, 2H), 4.31-4.18 (m, 4H), 4.10-4.03 (m, 4H), 3.65-3.57 (m,2H), 3.53-3.38 (m, 4H), 3.18 (ddd, J=16.3, 11.0, 2.1 Hz, 2H), 2.26-2.18(m, 2H), 1.75-1.66 (m, 4H), 1.55-1.46 (m, 4H), 1.44-1.35 (m, 4H),0.85-0.70 (m, 4H), −0.08 (s, 18H).

A mixture of compound 55 (65 mg, 0.054 mmol), (4-methoxyphenyl)boronicacid (18.12 mg, 0.119 mmol), and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)(PdCl₂(dppf), 1.983 mg, 2.71 μmol) was evacuated and backfilled with N₂,then THF (1084 μL) and tribasic potassium phosphate (542 μL, 0.5 Msolution in H₂O, 0.271 mmol) were added. The mixture was sparged with N₂for 5 min then stirred at RT for 1.5 h. The reaction was diluted withEtOAc (50 mL) and washed with sat. aq. NaHCO₃ (50 mL) and sat. aq. NaCl(50 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. Thecrude material was purified by flash chromatography (24 g silica gelwith 5 g prepacked load cartridge; linear gradient 0-100% EtOAc-hexanes)to provide compound 56a (50.3 mg, 83%) as a white foam. LC-MS m/z 1116[M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.42 (s, 2H), 7.40-7.36 (m, 4H),7.34-7.32 (m, 2H), 7.29 (s, 2H), 6.91-6.87 (m, 4H), 5.53 (d, J=10.1 Hz,2H), 4.76 (d, J=10.1 Hz, 2H), 4.65 (dd, J=10.5, 3.4 Hz, 2H), 4.32 (t,J=6.2 Hz, 4H), 4.19-4.06 (m, 4H), 3.98-3.91 (m, 2H), 3.83 (s, 6H),3.84-3.77 (m, 2H), 3.71 (td, J=9.6, 7.0 Hz, 2H), 3.15 (ddd, J=16.1,10.7, 2.1 Hz, 2H), 2.35 (quin, J=5.9 Hz, 2H), 1.85-1.77 (m, 4H),1.64-1.55 (m, 4H), 1.50-1.42 (m, 4H), 1.00 (ddd, J=9.5, 7.0, 2.2 Hz,4H), 0.04 (s, 18H).

To a −78° C. solution of compound 56a (50.3 mg, 0.045 mmol) in THF (1503μL) was added lithium triethylborohydride (225 μL, 1 M solution in THF,0.225 mmol) dropwise. The reaction mixture was stirred at −78° C. for 1h. The reaction mixture was diluted with H₂O (10 mL) and extracted withDCM (2×10 mL). The combined organic layers were dried over Na₂SO₄,filtered, and concentrated in vacuo. The residue was taken up in CHCl₃(1.0 mL) and EtOH (1.0 mL), then silica gel (0.50 g) and H₂O (0.50 mL)were added. The reaction was stirred at RT for 3 days. The mixture wasfiltered through CELITE™ (washed with CHCl₃) and the filtrate wasconcentrated in vacuo. The crude material was purified by preparativeHPLC (3 injections, each in 2 mL of DMSO; Phenomenex Luna C18 21.2×100mm; linear gradient 42-90% MeCN—H₂O with 0.1% v/v TFA over 12 min; 20mL/min; 220 nm detection). The product-containing fractions wereimmediately diluted with sat. aq. NaHCO₃ (100 mL) and extracted withCHCl₃ (2×50 mL). The combined organic layers were dried over MgSO₄,filtered, and concentrated in vacuo to provide dimer IIc-7 (6.0 mg, 15%)as a orange solid. LC-MS m/z 823 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.89(d, J=4.0 Hz, 2H), 7.54 (s, 2H), 7.40 (s, 2H), 7.37-7.33 (m, 4H),6.94-6.89 (m, 4H), 6.88 (s, 2H), 4.47-4.40 (m, 2H), 4.39-4.25 (m, 4H),4.22-4.16 (m, 2H), 4.14-4.05 (m, 2H), 3.84 (s, 6H), 3.59 (ddd, J=16.3,11.5, 1.9 Hz, 2H), 3.43-3.34 (m, 2H), 2.42-2.29 (m, 2H), 1.86-1.76 (m,4H), 1.64-1.55 (m, 4H), 1.49-1.43 (m, 4H).

Dimer IIc-8 was prepared analogously according to the syntheticprocedures described above for dimer IIc-7. The analytical data fordimer IIc-8 were: LC-MS m/z 960 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.88(d, J=4.0 Hz, 2H), 7.55-7.52 (m, 2H), 7.39-7.36 (m, 2H), 7.34-7.30 (m,4H), 6.94-6.90 (m, 4H), 6.89-6.87 (m, 2H), 4.45-4.38 (m, 2H), 4.37-4.25(m, 4H), 4.21-4.15 (m, 2H), 4.14-4.05 (m, 2H), 3.62-3.53 (m, 2H),3.42-3.33 (m, 2H), 3.29-3.22 (m, 8H), 2.62-2.56 (m, 8H), 2.37 (s, 6H),2.38-2.30 (m, 2H), 1.84-1.76 (m, 4H), 1.62-1.52 (m, 4H), 1.48-1.40 (m,4H).

Example 7 Dimers IIc-9, IIc-10, IIc-11, and IId-1

This example pertains to FIG. 7 and the synthesis of dimers IIc-9,IIc-10, IIc-11 and IId-1.

A mixture of compound 55 (0.51g, 0.425 mmol), (4-aminopehnyl)boronicacid (58 mg, 0.425mmol), and PcCl₂(dppf) (16 mg, 21 μmol) was evacuatedand backfilled with N₂. THF (8.5 mL) and tribasic potassium phosphate(4.25 mL, 0.5 M solution in H₂O, 2.126 mmol) were added. The mixture wassparged with N₂ for 5 min then stirred at RT for 1 h. The reactionmixture was diluted with DCM (30 mL), washed with sat. aq. NaCl (30 mL),dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudematerial was purified by flash chromatography (40 g column; lineargradient 0-100% EtOAc-hexanes) to provide compound 57b (205 mg, 42%) asa yellow foam. LC-MS m/z 1142 [M+H]⁺.

A mixture of compound 57b (70 mg, 0.061 mmol), (4-methoxyphenyl)boronicacid (12.1 mg, 0.080 mmol), and PcCl₂(dppf) (2.24 mg, 3.1 μmol) wasevacuated and backfilled with N₂. THF (1226 μL) and tribasic potassiumphosphate (613 μL, 0.5 M solution in H₂O, 0.306 mmol) were added. Themixture was sparged with N₂ for 5 min then stirred at RT for 30 min. Thereaction was diluted with EtOAc (30 mL), washed with sat. aq. NaCl (30mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudematerial was purified by flash chromatography (40 g column; lineargradient 0-100% EtOAc-hexanes) to provide compound 58a (60 mg, 89%) as ayellow foam. LC-MS m/z 1100.8 [M+H]⁺.

Alternatively to using a boronic acid, a Grignard reagent can be used,as illustrated by the following synthesis of compound 58d from compound57b: To a −30° C. solution of compound 57b (112 mg, 0.098 mmol) andiron(III) acetylacetonate (3.46 mg, 9.80 μmol) in THF (1334 μL) and NMP(66.7 μL) was added methylmagnesium bromide (131 μL, 3.0 M solution inEt₂O, 0.392 mmol), dropwise slowly. The reaction was stirred at −30° C.for 10 min, then it was quenched by the addition of sat. aq. NH₄Cl (30mL) and extracted with EtOAc (2×30 mL). The combined organic layers werewashed with sat. aq. NaCl (30 mL), dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude material was purified by flashchromatography (12 g silica gel with 5 g prepacked load cartridge;linear gradient 0-100% EtOAc—CH₂Cl) to provide compound 58d (50 mg, 51%)as a yellow foam. LC-MS m/z 1008 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ7.42-7.38 (m, 2H), 7.29-7.24 (m, 5H), 6.70-6.64 (m, 3H), 5.55-5.48 (m,2H), 4.77-4.71 (m, 2H), 4.62 (dd, J=10.6, 3.2 Hz, 1H), 4.48 (dd, J=10.4,3.3 Hz, 1H), 4.35-4.27 (m, 4H), 4.18-4.05 (m, 4H), 3.95-3.88 (m, 1H),3.84-3.64 (m, 6H), 3.49-3.42 (m, 1H), 3.12 (ddd, J=16.1, 10.5, 2.0 Hz,1H), 2.83-2.72 (m, 1H), 2.34 (quin, J=6.0 Hz, 2H), 1.84 (d, J=1.1 Hz,3H), 1.83-1.76 (m, 4H), 1.63-1.56 (m, 4H), 1.49-1.43 (m, 4H), 0.99 (ddd,J=9.6, 6.9, 2.4 Hz, 4H), 0.04 (s, 18H).

To a −78° C. solution of the crude compound 58a (30 mg, 0.027 mmol) inTHF (1 mL) was added lithium triethylborohydride (273 μL, 1 M solutionin THF, 0.273 mmol) dropwise. The reaction was stirred at −78° C. for 1h. The reaction was diluted with brine and extracted with EtOAc (3×).The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude material was taken up in EtOH/THF (1:1,2 mL) and aq. formic acid (0.055, 1 mL). The resulting solution wasstirred at RT for 2h before it was neutralized with aq. NaHCO_(3.) Theresulting mixture was extracted with chloroform (3×). The combinedorganic layers were dried over Na₂SO₄, filtered, and concentrated invacuo. The crude product was then purified by flash chromatography (12 gcolumn, 0-10% MeOH/DCM) to give dimer IIc-11 (11 mg, 45%). LC-MS m/z808.4 [M+H]⁺.

Dimers IIc-9, IIc-10, and IId-1 were analogously prepared according tothe synthetic procedures described for above for dimer IIc-11. Theiranalytical data is provided below.

Dimer IIc-9: LC-MS m/z 876 [M+H]⁺; ¹⁻E1 NMR (400 MHz, CDCl₃) δ 7.90-7.86(m, 2H), 7.56-7.52 (m, 2H), 7.39-7.36 (m, 1H), 7.35-7.33 (m, 1H), 7.31(d, J=8.6 Hz, 2H), 7.22 (d, J=8.6 Hz, 2H), 6.92 (d, J=9.0 Hz, 2H), 6.88(s, 2H), 6.68 (d, J=8.6 Hz, 2H), 4.45-4.37 (m, 2H), 4.36-4.25 (m, 4H),4.22-4.15 (m, 2H), 4.13-4.06 (m, 2H), 3.79-3.69 (m, 2H), 3.63-3.52 (m,2H), 3.41-3.32 (m, 2H), 3.31-3.23 (m, 4H), 2.65-2.57 (m, 4H), 2.38 (s,3H), 2.41-2.30 (m, 2H), 1.84-1.76 (m, 4H), 1.62-1.54 (m, 4H), 1.49-1.40(m, 4H).

Dimer IIc-10: LC-MS m/z 946 [M+H]⁻; ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d,J=4.0 Hz, 2H), 7.56-7.51 (m, 2H), 7.39-7.36 (m, 2H), 7.35-7.29 (m, 4H),6.94-6.90 (m, 4H), 6.89-6.86 (m, 2H), 4.45-4.38 (m, 2H), 4.37-4.24 (m,4H), 4.22-4.15 (m, 2H), 4.13-4.05 (m, 2H), 3.62-3.52 (m, 2H), 3.42-3.33(m, 2H), 3.29-3.22 (m, 4H), 3.22-3.15 (m, 4H), 3.07-3.02 (m, 4H),2.61-2.55 (m, 4H), 2.39-2.29 (m, 6H), 1.84-1.76 (m, 4H), 1.64-1.51 (m,4H), 1.48-1.40 (m, 4H).

Dimer IId-1: LC-MS 716 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J=4.0Hz, 1H), 7.80 (d, J=4.0 Hz, 1H), 7.55-7.50 (m, 2H), 7.35-7.32 (m, 1H),7.24-7.20 (m, 2H), 6.88-6.84 (m, 2H), 6.76-6.73 (m, 1H), 6.70-6.66 (m,2H), 4.44-4.36 (m, 1H), 4.35-4.22 (m, 5H), 4.22-4.14 (m, 2H), 4.13-4.05(m, 2H), 3.84-3.69 (m, 2H), 3.61-3.48 (m, 1H), 3.40-3.31 (m, 1H),3.23-3.11 (m, 1H), 3.00-2.91 (m, 1H), 2.34 (quin, J=6.1 Hz, 2H), 1.84(d, J=1.1 Hz, 3H), 1.82-1.75 (m, 4H), 1.63-1.53 (m, 4H), 1.48-1.40 (m,4H).

Example 8 Dimer IIb-6

This example pertains to FIG. 8 and the synthesis of dimer IIb-6.

To a RT solution of compound 44 (1.006 g, 1.834 mmol) in DMF (12.23 mL)was added N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU, 1.743 g, 4.59 mmol) followed by (S)-ethyl7-nitro-1,2,3,4-tetrahydroisoquinoline-3-carboxylate 44b (0.459 g, 1.834mmol) portionwise, and then N,N-diisopropylethylamine (DIEA, 1.917 mL,11.00 mmol) dropwise. The resulting clear brown solution was stirred atRT for 30 min, then (S)-benzyl1,2,3,4-tetrahydroisoquinoline-3-carboxylate 4-methylbenzenesulfonate44c (0.806 g, 1.834 mmol) was added. The reaction was stirred at RT foran additional 1 h. The reaction mixture was slowly added to a stirredflask of H₂O (125 mL) at 0° C. and the resulting precipitate wascollected by vacuum filtration (washed with H₂O). The solids weredissolved in EtOAc (200 mL), washed with sat. aq. NaCl (150 mL), driedover Na₂SO₄, filtered, and concentrated in vacuo. The crude material waspurified by flash chromatography (220 g Redi Sep Gold silica gel with 25g prepacked load cartridge; linear gradient 0-100% EtOAc-hexanes) toprovide compound 67 (538 mg, 29%) as a brown foam. LC-MS m/z 1030[M+H]⁺.

To a RT solution of compound 67 (538 mg, 0.522 mmol) in MeOH (3264 μL)and THF (3264 μL) was added NH_(4Cl) (559 mg, 10.45 mmol), zinc dust(683 mg, 10.45 mmol), and HOAc (1 drop). The resulting suspension wasstirred at 60° C. for 18 h. The reaction was cooled to RT and filteredthrough CELITE™ (washed with EtOAc, DCM, MeOH, and %5 v/v Et₃N in DCM).The filtrate was concentrated in vacuo. The crude material was taken upin DCM (200 mL), washed with sat. aq. NaHCO₃ (200 mL) and H₂O (200 mL),dried over Na₂SO₄, filtered, and concentrated in vacuo to providecompound 68 (250 mg, 61%) as an orange solid. LC-MS m/z 786 [M+H]⁺.

To a 0° C. solution of compound 68 (250 mg, 0.318 mmol) in DMF (3181 μL)was added NaH (63.6 mg, 60% w/w in mineral oil, 1.591 mmol). Thereaction was stirred at 0° C. for 30 min, then SEM-Cl (226 μL, 1.272mmol) was added. The reaction was stirred at 0° C. for 30 min. Thereaction was quenched by the addition of sat. aq. NH₄Cl, then it waswarmed to RT, diluted with EtOAc (100 mL), washed with H₂O (100 mL) andsat. aq. NaCl (100 mL), dried over Na₂SO₄, filtered, and concentrated invacuo. The crude material was purified by flash chromatography (40 gsilica gel with 5 g prepacked load cartridge; linear gradient 0-10%MeOH—CH₂Cl₂) to provide compound 69 (289 mg, 87%) as a yellow foam.LC-MS m/z 1047 [M+H]⁺.

To a −78° C. solution of compound 69 (43.9 mg, 0.042 mmol) in THF (1398μL) was added lithium triethylborohydride (210 μL, 1 M solution in THF,0.210 mmol) dropwise. The reaction was stirred at −78° C. for 1.5 h. Thereaction was diluted with H₂O (10 mL) and extracted with CHCl₃ (2×10mL). The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue was taken up in CHCl₃ (1 mL) and EtOH(1 mL), then silica gel (0.5 g) and H₂O (0.5 mL) were added. Thereaction was stirred at RT for 2 days. The mixture was filtered throughCELITE™ (washed with acetone, CHCl₃, and 10% MeOH—CHCl₃) and thefiltrate was concentrated in vacuo. This material was taken up in CHCl₃and H₂O and the layers were separated. The aqueous layer was extractedwith CHCl₃, dried over Na₂SO₄, filtered, and concentrated in vacuo. Thecrude material was purified by preparative HPLC (2 injections, each in 2mL of 1:1 MeCN-DMSO; Phenomenex Luna C18 21.2×100 mm; linear gradient26-90% MeCN—H₂O with 0.1% v/v TFA over 12 min; 20 mL/min; 220 nmdetection). The product-containing fractions were immediately dilutedwith sat. aq. NaHCO₃ (100 mL) and extracted with CHCl₃ (2×50 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo to provide dimer IIb-6 (6.6 mg, 19%) as anoff-white solid. LC-MS m/z 754 [M+H]⁺; ^(i)H NMR (400 MHz, CDCl₃) δ7.54-7.52 (m, 2H), 7.49-7.45 (m, 2H), 7.39-7.29 (m, 4H), 7.13 (d, J=8.1Hz, 1H), 6.86-6.83 (m, 2H), 6.66-6.62 (m, 2H), 5.01 (d, J=15.6 Hz, 1H),4.90 (d, J=15.6 Hz, 1H), 4.56 (d, J=15.6 Hz, 1H), 4.44 (d, J=15.4 Hz,1H), 4.35-4.23 (m, 4H), 4.22-4.15 (m, 2H), 4.08 (dt, J=9.6, 4.9 Hz, 2H),3.98-3.93 (m, 1H), 3.92-3.87 (m, 1H), 3.78-3.65 (m, 2H), 3.31-3.24 (m,1H), 3.20-3.12 (m, 2H), 3.05-2.99 (m, 1H), 2.33 (quin, J=6.1 Hz, 2H),1.84-1.74 (m, 4H), 1.62-1.50 (m, 4H), 1.48-1.39 (m, 4H).

Example 9 Dimer IId-2

This example pertains to FIG. 9 and the synthesis of dimer IId-2.

Following the reaction scheme shown in FIG. 9, dimer IId-2 wassynthesized analogously to the procedures of the previous example. LC-MSm/z 704 [M+H]⁺.

Example 10 Dimers IId-3 and IId-4

This example pertains to FIG. 10 and the synthesis of dimers IId-3 andIId-4.

To a −78° C. suspension of dimer IIc-8 (46.2 mg, 0.048 mmol) in THF (482μL) was added LiBHEt₃ (48.2 μL, 1 M solution in THF, 0.048 mmol),dropwise. The reaction mixture was stirred at −78° C. for 15 min. Thenit was quenched by the addition of H₂O, warmed to RT, diluted with sat.aq. NaHCO₃ (25 mL) and H₂O (25 mL), and extracted with 10% MeOH—CHCl₃(2×50 mL). The combined organic layers were dried over Na₂SO₄, filtered,and concentrated in vacuo. The crude material was purified bypreparative HPLC (4 1-mL injections in DMSO; Phenomenex Luna C1821.2×100 mm; linear gradient 0-50% MeCN—H₂O w/0.05% v/v HCO₂H over 25min; 20 mL/min; 220 nm detection). The fractions containing the mono-and bis-reduced products were separately lyophilized then repurified bypreparative HPLC to provide dimer IId-3 (2.7 mg, 6%) as a light yellowsolid and dimer IId-4 (1.24 mg, 3%), also as a light yellow solid. Theiranalytical data is presented below.

Dimer IId-3: LC-MS m/z 961 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d,J=4.0 Hz, 1H), 7.56 (s, 1H), 7.54-7.52 (m, 1H), 7.52-7.50 (m, 1H),7.39-7.37 (m, 1H), 7.34-7.29 (m, 4H), 6.94-6.86 (m, 5H), 6.13 (s, 1H),4.41 (dt, J=11.1, 4.7 Hz, 1H), 4.37-4.22 (m, 5H), 4.18 (dt, J=9.8, 5.1Hz, 1H), 4.13-3.96 (m, 3H), 3.62-3.52 (m, 3H), 3.41-3.32 (m, 2H),3.31-3.24 (m, 8H), 2.75-2.69 (m, 1H), 2.70-2.64 (m, 8H), 2.41 (s, 6H),2.35-2.28 (m, 2H), 1.84-1.70 (m, 4H), 1.62-1.51 (m, 4H), 1.47-1.38 (m,4H).

Dimer IId-4: LC-MS m/z 963 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.56 (s,2H), 7.51-7.50 (m, 2H), 7.30-7.26 (m, 4H), 6.90 (d, J=8.8 Hz, 4H), 6.10(s, 2H), 4.36-4.29 (m, 2H), 4.24-4.17 (m, 4H), 4.10-3.97 (m, 4H),3.59-3.50 (m, 4H), 3.40-3.32 (m, 2H), 3.27-3.21 (m, 8H), 2.72 (dd,J=16.0, 3.4 Hz, 2H), 2.62-2.55 (m, 8H), 2.36 (s, 6H), 2.34-2.26 (m, 2H),1.77-1.68 (m, 4H), 1.64-1.50 (m, 4H), 1.43-1.36 (m, 4H).

Example 11 Dimer-Linker IIIa-1

This example pertains to FIG. 11 and the synthesis of dimer-linkerIIIa-1.

To a 0° C. mixture of dipeptide 69a (70.4 mg, 0.142 mmol) and HATU (53.9mg, 0.142 mmol) was added DMF (945 μL). The mixture was stirred at 0° C.for 10 min and 2,6-lutidine (22.02 μL, 0.189 mmol) was added. Thismixture was added dropwise to compound 69 (98.9 mg, 0.095 mmol) in avial at 0° C. The reaction was allowed to warm to RT as it was stirredfor 22 h. Then it was added dropwise to a stirred flask of H₂O (20 mL)in a 0° C. bath. The precipitate was collected by vacuum filtration(washed with H₂O), taken up in DCM (50 mL), and washed with H₂O (50 mL).The aqueous layer was extracted with DCM (50 mL). The combined organiclayers were dried over Na₂SO₄, filtered, and concentrated in vacuo. Thecrude material was purified by flash chromatography (24 g silica gelwith 5 g prepacked load cartridge; linear gradient 0-10% MeOH-DCM). Themixed fractions were repurified by flash chromatography (24 g RediSepGold silica gel with 5 g prepacked load cartridge; linear gradient 0-10%MeOH—CH₂Cl) and the products of the two columns were combined to providecompound 70 (68.8 mg, 48%). LC-MS m/z 1525 [M+H]⁺.

To a RT solution of compound 70 (39.9 mg, 0.026 mmol) in DMF (1047 μL)was added silica-supported piperazine (575 mg, 0.91 mmol/g loading,0.523 mmol). The suspension was stirred at RT for 18 h, then it wasfiltered (washed with ˜2 mL DMF), and the filtrate was concentrated invacuo. This crude compound 71 was combined with crude compound 71 fromanother batch (0.019 mmol scale) and used without further purification.LC-MS m/z 1303 [M+H]⁺.

To a −78° C. solution of crude compound 71 in THF (1356 μL) was addedLiBHEt₃ (203 μL, 1 M solution in THF, 0.203 mmol), dropwise. Thereaction was stirred at −78° C. for 1.5 h, then it was diluted with sat.aq. NaCl (50 mL) and extracted with DCM (2×50 mL). The combined organiclayers were dried over Na₂SO₄, filtered, and concentrated in vacuo. Theresidue was taken up in CHCl₃ (1 mL) and EtOH (1 mL), then silica gel(0.5 g) and H₂O (0.5 mL) were added. The reaction mixture was stirred atRT for 2 days, then it was filtered through CELITE™ (washed with 10%EtOH—CHCl₃) and the filtrate was concentrated in vacuo. The crudematerial was purified by preparative HPLC (2 injections, each in 2 mLDMSO; Phenomenex Luna C18 21.2×100 mm; linear gradient 18-90% MeCN—H₂Owith 0.1% v/v TFA over 12 min; 20 mL/min; 220 nm detection). Theproduct-containing fractions were filtered under gravity through an SPEcartridge packed with PL-HCO₃ MP resin (Agilent, 500 mg, 1.8 mmol/gloading) (washed with3 mL of 1:1 MeCN—H₂O), and the eluent waslyophilized to provide compound 72 (4.9 mg, 12%) as a white solid. LC-MSm/z 1011 [M+H]⁺.

To a RT solution of compound 72 (4.9 mg, 4.85 μmol) in DMSO (129 μL) wasadded a solution of compound 71a (6.69 mg, 9.70 μmol) in DMSO (64.7followed by 2,6-lutidine (1.130 μL, 9.70 μmol). The clear colorlesssolution was stirred at RT for 4 h. The reaction was purified bypreparative HPLC (1 injection; Phenomenex Luna C18 21.2×100 mm; lineargradient 18-90% MeCN—H₂O with 0.1% v/v TFA over 12 min; 20 mL/min; 220nm detection). The product-containing fraction was filtered undergravity through an SPE cartridge packed with PL-HCO₃ MP resin (Agilent,200 mg, 1.8 mmol/g loading) (washed with 2 mL of 1:1 MeCN—H₂O), and theeluent was lyophilized to provide dimer-linker compound IIIa-1 (2.4 mg,31%) as a white solid. LC-MS m/z 1585 [M+H]⁺.

Example 12 Dimer-Linkers IIIa-2, IIIa-3, and IIIa-4

This example pertains to FIG. 12 and the synthesis of dimer-linkersIIIa-2, IIIa-3, and IIIa-4.

To a RT solution of dimer IIb-6 (7 mg, 9.29 μmol) in DMF (93 μL) wasadded a solution of compound 72a (12.47 mg, 0.011 mmol) in DMF (93 μL),followed by DIEA (4.85 μL, 0.028 mmol). The clear orange solution wasstirred at RT for 15 mi; then 1-hydroxy-7-azabenzotriazole (1.517 mg,0.011 mmol) was added. The reaction mixturewas stirred at RT for 29 h,then diluted with DMSO and purified by preparative HPLC (1 injection;Phenomenex Luna C18 21.2×100 mm; linear gradient 18-90% MeCN—H₂O with0.1% v/v TFA over 15 min; 20 mL/min; 220 nm detection). Theproduct-containing fraction was filtered under gravity through an SPEcartridge packed with PL-HCO₃ MP resin (Agilent, 200 mg, 1.8 mmol/gloading) (washed with 2 mL of 1:1 MeCN—H2O), and the eluent waslyophilized to provide dimer-linker IIIa-2 (4.43 mg, 28%) as a whitesolid. LC-MS m/z 1734 [M+H]+.

Dimer-linkers IIIa-3 and IIIa-4 were analogously prepared. Dimer-linkerIIIa-3: LC-MS m/z 1684 [M+H]⁺. Dimer-linker IIIa-4: LC-MS m/z 1925[M+H]⁺.

Example 13 Dimer-Linker IIIa-11

This example pertains to FIGS. 13A-13B and the synthesis of dimer-linkerIIIa-11.

To a 0° C. solution of compound 44e (2.065 g, 8.09 mmol) in DMF (25.7mL) was added compound 44 (2.114 g, 3.85 mmol), followed by DIEA (5.37mL, 30.8 mmol) and HATU (3.22 g, 8.48 mmol), portionwise. The reactionwas stirred at 0° C. for 5 min and RT for 1 h. Then it was slowly addedto a stirred flask of H₂O (200 mL) in a 0° C. bath. The resultingprecipitate was collected by vacuum filtration (H₂O wash), dissolved inEtOAc (200 mL), washed with sat. aq. NaCl (200 mL), dried over Na₂SO₄,filtered, and concentrated in vacuo. The crude material was purified byflash chromatography (40 g RediSep Gold silica gel with 25 g prepackedload cartridge; linear gradient 0-100% EtOAc—CH₂Cl₂) to provide compound76 (3.1 g, quantitative) as an orange film. LC-MS m/z 795 [M+H]⁻.

To a 0° C. solution of compound 76 (3.1 g, 3.90 mmol) in THF (39.0 mL)was added LiBH₄ (5.85 mL, 2.0 M solution in THF, 11.70 mmol), dropwise.The reaction was stirred at 0° C. for 30 min, then was allowed to warmto RT and stirred for an additional 3 h. The reaction was cooled to 0°C. and quenched by the addition of 1 M aq. HCl (100 mL), diluted withH₂O (400 mL), and extracted with 10% MeOH-EtOAc (400 mL) and EtOAc (400mL). The combined organic layers were washed with sat. aq. NaCl (200mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudeproduct 77 was used without further purification. LC-MS MS m/z 739[M+H]⁺.

To a 0° C. solution of the crude product 77 in DCM (77 mL) was addedNEt₃ (1.607 mL, 11.53 mmol), followed by acetyl chloride (0.713 mL, 9.99mmol). The reaction was allowed to warm to RT as it was stirred for 20h, diluted with DCM (400 mL), washed with H₂O (400 mL), dried overNa₂SO₄, filtered, and concentrated in vacuo. The crude material waspurified by flash chromatography (80 g silica gel with 25 g prepackedload cartridge; linear gradient 0-100% EtOAc—hexanes) to providecompound 78 (1.59 g, 50%) as a white foam. LC-MS m/z 823 [M+H]⁺.

To a RT solution of compound 78 (1.59 g, 1.932 mmol) in EtOH (61.8 mL)was added AcOH (15.46 mL), followed by zinc dust (3.79 g, 58.0 mmol).The reaction was stirred at reflux for 1 h, then it was cooled to RT andfiltered through CELITE™ (washed with 400 mL DCM). The filtrate waswashed with H₂O (400 mL), sat. aq. NaHCO₃ (400 mL), and H₂O (400 mL),dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudematerial was purified by flash chromatography (40 g silica gel with 2.5g prepacked load cartridge; linear gradient 0-10% MeOH—CH₂Cl₂). Themixed fractions were repurified by flash chromatography (12 g silica gelwith 2.5 g prepacked load cartridge; linear gradient 0-10% MeOH-DCM) andthe products from both columns were combined to provide compound 79(1.162 g, 79%) as a white foam. LC-MS m/z 763 [M+H]⁺; ¹H NMR (400 MHz,CDCl₃) δ 6.80 (s, 2H), 6.27 (s, 2H), 5.04-5.00 (m, 2H), 4.99-4.95 (m,2H), 4.86-4.75 (m, 2H), 4.37 (br s, 4H), 4.24-4.08 (m, 12H), 3.94 (t,J=5.5 Hz, 4H), 2.83-2.72 (m, 2H), 2.50-2.42 (m, 2H), 2.31 (quin, J=6.4Hz, 2H), 2.04 (s, 6H), 1.76-1.65 (m, 4H), 1.61-1.52 (m, 4H), 1.44-1.37(m, 4H).

To a 0° C. solution of triphosgene (117 mg, 0.396 mmol) and NEt₃ (487μL, 3.49 mmol) in THF (3695 μL) was added a solution of compound 79 (444mg, 0.582 mmol) in THF (3695 μL), dropwise. The cloudy mixture wasstirred at 0° C. for 10 min, then a suspension of compound 79a (231 mg,0.611 mmol) in THF (5543 μL) was added dropwise, followed by allylalcohol (41.7 μL, 0.611 mmol). The suspension was stirred at 0° C. for 1h, then it was allowed to warm to RT as it was stirred for 5 h. Thereaction was diluted with H₂O (125 mL) and extracted with CH₂Cl₂ (2×125mL). The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude material was purified by flashchromatography (80 g RediSep Gold silica gel with 5 g prepacked loadcartridge; linear gradient 0-10% MeOH-DCM) to provide compound 80 (214mg, 29%) as an off-white solid. LC-MS m/z 1250 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d₆) δ 9.98 (s, 1H), 9.13-9.02 (m, 2H), 8.14 (d, J=6.8 Hz, 1H), 7.58(d, J=8.6 Hz, 2H), 7.31 (d, J=8.7 Hz, 2H), 7.23 (d, J=8.7 Hz, 1H),7.20-7.12 (m, 2H), 6.99-6.89 (m, 2H), 5.98-5.85 (m, 2H), 5.36-5.26 (m,2H), 5.22-5.13 (m, 2H), 5.06-4.93 (m, 6H), 4.62-4.50 (m, 4H), 4.50-4.46(m, 2H), 4.45-4.38 (m, 1H), 4.22-3.80 (m, 17H), 2.78-2.67 (m, 2H),2.44-2.35 (m, 2H), 2.21-2.13 (m, 2H), 2.04-1.90 (m, 7H), 1.69-1.62 (m,4H), 1.54-1.46 (m, 4H), 1.40-1.35 (m, 4H), 1.30 (d, J=7.0 Hz, 3H), 0.88(d, J=6.8 Hz, 3H), 0.83 (d, J=6.6 Hz, 3H).

To a RT solution of compound 80 (213 mg, 0.170 mmol) in MeOH (3097 μL)was added H₂O (310 μL) and K₂CO₃ (118 mg, 0.852 mmol). The reaction wasstirred at RT for 1 h, diluted with H₂O (100 mL), and extracted withEtOAc (2×100 mL). The combined organic layers were washed with sat. aq.NaCl (100 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo.The crude material was purified by flash chromatography (40 g silica gelwith 5 g prepacked load cartridge; linear gradient 0-10% MeOH—CH₂Cl₂) toprovide compound 81 (159 mg, 80%) as a white solid. LC-MS m/z 1166[M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.99 (s, 1H), 9.17-8.86 (m, 2H),8.15 (d, J=7.0 Hz, 1H), 7.58 (d, J=8.6 Hz, 2H), 7.32 (d, J=8.7 Hz, 2H),7.27-7.16 (m, 3H), 7.03-6.96 (m, 2H), 5.98-5.85 (m, 2H), 5.35-5.26 (m,2H), 5.23-5.14 (m, 2H), 5.07-4.76 (m, 8H), 4.56-4.52 (m, 2H), 4.50-4.46(m, 2H), 4.45-4.39 (m, 1H), 4.37-4.30 (m, 1H), 4.18-4.11 (m, 4H),4.08-3.80 (m, 10H), 3.57-3.46 (m, 1H), 3.39-3.26 (m, 2H), 3.22-3.03 (m,1H), 2.65-2.46 (m, 4H), 2.21-2.12 (m, 2H), 2.03-1.93 (m, 1H), 1.70-1.61(m, 4H), 1.55-1.46 (m, 4H), 1.42-1.34 (m, 4H), 1.30 (d, J=7.0 Hz, 3H),0.88 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H).

To a RT solution of compound 81 (157 mg, 0.135 mmol) in CH₂Cl₂ (3365 μL)was added Dess-Martin periodinane (DMP, 120 mg, 0.283 mmol). Thereaction was stirred at RT for 4 h, diluted with sat. aq. NaHCO₃ (50mL), and extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudematerial was purified by flash chromatography (40 g silica gel with 5 gprepacked load cartridge; linear gradient 0-10% MeOH—CH₂Cl₂). The mixedfractions were repurified by flash chromatography (40 g RediSep silicagel with 5 g prepacked load cartridge; linear gradient 0-10%MeOH—CH₂Cl₂) and the products from both columns were combined to providecompound 82 (115 mg, 74%) as a white solid. LC-MS m/z 1162 [M+H]⁺.

To a RT suspension of compound 82 (96.5 mg, 0.083 mmol) in DCM (1661 μL)was added morpholine (36.5 μL, 0.415 mmol) andtetrakis(triphenylphosphine)palladium(O) (4.80 mg, 4.15 μmol). Thereaction was stirred at RT for 2 h, was concentrated under a stream ofN₂, and purified by flash chromatography (12 g silica gel; lineargradient 0-10% MeOH-DCM). The mixed fractions were repurified by flashchromatography (12 g silica gel; linear gradient 0-10% MeOH—CH₂Cl₂) andthe products from both columns were combined to provide compound 83(54.9 mg, 68%). LC-MS m/z 976 [M+H]⁺.

To a RT solution of compound 83 (42.3 mg, 0.043 mmol) in DMF (867 μL)was added 2,6-lutidine (15.14 μL, 0.130 mmol), followed by compound 83a(20.04 mg, 0.065 mmol). The reaction was stirred at RT for 2 days,diluted with DMSO, and purified by preparative HPLC (5 1-mL injections;Phenomenex Luna C18 21.2×100 mm; linear gradient 20-80% MeCN—H₂O with0.05% v/v HCO₂H over 25 min; 20 mL/min; 220 nm detection). Theproduct-containing fractions were lyophilized and purified by flashchromatography (12 g RediSep Gold silica gel; linear gradient 0-20%MeOH-DCM) to provide dimer-linker IIIa-11 (3.67 mg, 7%) as an off-whitesolid. LC-MS m/z 1170 [M+H]⁺.

Example 14 Dimer-Linker 111a-5

This example pertains to FIG. 14 and the synthesis of dimer-linkerIIIa-5.

A mixture of compound 57a (82.5 mg, 0.067 mmol), compound 84 (45.3 mg,0.074 mmol), PdCl₂(dppf) (2.463 mg, 3.37 μmol), and Na₂CO₃ (35.7 mg,0.337 mmol) was evacuated and backfilled with N₂. THF (898 μL) and H₂O(449 μL) were added. The mixture was sparged with N₂ for 5 min andstirred at RT for 30 min. The reaction mixture was diluted H₂O (50 mL)and extracted with DCM (2×50 mL). The combined organic layers were driedover Na₂SO₄, filtered, and concentrated in vacuo. The crude material waspurified by flash chromatography (24 g silica gel with 5 g prepackedload cartridge; linear gradient 0-20% MeOH-DCM) to provide compound 85(98.9 mg, 94%) as an orange film. LC-MS m/z 1561 [M+H]⁺.

To a −78° C. solution of compound 85 (98.9 mg, 0.063 mmol) in THF (2112μL) was added LiBHEt₃ (317 μL, 1 M solution in THF, 0.317 mmol),dropwise. The reaction mixture was stirred at −78° C. for 1 h, dilutedwith H₂O (50 mL) and extracted with CHCl₃ (50 mL) and 10% MeOH—CHCl₃ (50mL). The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue was taken up in a mixture of THF(4217 μL), MeCN w/0.05% v/v HCO₂H (2109 μL), and H₂O w/0.05% v/v HCO₂H(2109 μL), and stirred at RT for 1 h. The reaction was quenched by theaddition of sat. aq. NaHCO₃ (50 mL) and extracted with CHCl₃ (2×50 mL).The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude material was purified by flashchromatography (24 g basic alumina; linear gradient 0-20% MeOH—CHCl₃) toprovide compound 86 (54.6 mg, 68%) as an orange solid. LC-MS m/z 1286[M+18]⁺.

To a RT solution of compound 86 (29.0 mg, 0.023 mmol) in THF (457 μL)was added piperidine (45.3 μL, 0.457 mmol). The clear orange solutionwas stirred at RT for 45 min and then concentrated in vacuo. The crudematerial was taken up in a mixture of MeCN (2 mL) and MeOH (2 mL) andwashed with heptane (4×2 mL). The MeCN—MeOH layer was concentrated invacuo. The residue was taken up in CHCl₃ and concentrated (2×), to givecompound 87, which was used without further purification. LC-MS m/z 1065[M+H]⁺.

To a RT solution of crude 87 and compound 71a (23.65 mg, 0.034 mmol) inDMSO (457 μL) was added 2,6-lutidine (6.66 μL, 0.057 mmol). The clearyellow solution was stirred at RT for 1.5 h, diluted with DMSO, andpurified by preparative HPLC (3 1-mL injections; Phenomenex Luna C1821.2×100 mm; linear gradient 20-60% MeCN—H₂O w/0.05% v/v HCO₂H over 25min; 20 mL/min; 220 nm detection). The product-containing fractions werelyophilized to provide dimer-linker IIIa-5 (6.97 mg, 19%) as a lightyellow solid. LC-MS m/z 1621 [M+H]⁺.

Example 15 Dimer-Linker IIIa-6

This example pertains to FIG. 15 and the synthesis of dimer-linkerIIIa-6.

To a 0° C. solution of compound 58d (50.0 mg, 0.050 mmol) and compound88 (24.42 mg, 0.060 mmol) in DMF (496 μL) was added HATU (22.62 mg,0.060 mmol), followed by 2,6-lutidine (14.44 μL, 0.124 mmol). Thereaction mixture was stirred at 0° C. for 10 min and RT for 2 h andadded dropwise to a stirred flask of H₂O (20 mL) in a 0° C. bath. Theresulting precipitate was collected by vacuum filtration (washed withH₂O), then taken up in DCM (50 mL) and sat. aq. NaHCO₃ (50 mL). Thelayers were separated and the aqueous layer was extracted with DCM (50mL). The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude product was purified by flashchromatography (12 g silica gel; linear gradient 0-10% MeOH-DCM) toprovide compound 89 (61.5 mg, 89%) as a yellow solid. LC-MS m/z 1401[M+H]⁺.

To a −78° C. solution of compound 89 (61.5 mg, 0.044 mmol) in THF (1463μL) was added LiBHEt₃ (220 μL, 1 M solution in THF, 0.220 mmol),dropwise. The reaction mixture was stirred at −78° C. for 1 h, quenchedby the addition of H₂O, warmed to RT, diluted with a mixture of sat. aq.NaHCO₃ (25 mL) and H₂O (25 mL), and extracted with 10% MeOH—CHCl₃ (2×50mL). The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. This residue was taken up in a mixture of THF(5867 μL), EtOH (5867 μL), and H₂O w/0.05% v/v HCO₂H (2933 μL) andstirred at RT for 2 h. The reaction was diluted with sat. aq. NaHCO₃ (50mL) and extracted with CHCl₃ (50 mL), followed by 10% MeOH—CHC₃ (50 mL).The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude product was purified by flashchromatography (12 g silica gel; linear gradient 0-20% MeOH-DCM) toprovide compound 90 (49 mg, quant.) as a yellow solid. LC-MS m/z 1109[M+H]⁺.

To a RT solution of compound 90 (49 mg, 0.044 mmol) in THF (884 μL) wasadded piperidine (88 μL, 0.884 mmol). The clear orange solution wasstirred at RT for 1 h and concentrated in vacuo. The crude product wastaken up in a mixture of MeCN (2 mL) and MeOH (2 mL) and washed withheptane (4×2 mL). The MeCN—MeOH layer was concentrated in vacuo. Thisproduct 91 was taken up in CHCl₃ and concentrated (2×) and then usedwithout further purification. LC-MS m/z 887 [M+H]⁺.

To a RT solution of crude product 91 and compound 71a (22.76 mg, 0.033mmol) in

DMSO (440 μL) was added 2,6-lutidine (6.41 μL, 0.055 mmol). The clear,yellow solution was stirred at RT for 30 min. An additional solution ofcompound 71a (3.2 0.027 mmol) in DMSO (0.220 mL) was added, and thereaction mixture was stirred for an additional 1.5 h, diluted with DMSOand purified by preparative HPLC (3 1-mL injections; Phenomenex Luna C1821.2×100 mm; linear gradient 20-60% MeCN—H₂O w/0.05% v/v HCO₂H over 25min; 20 mL/min; 220 nm detection). The product-containing fractions werelyophilized to provide dimer-linker IIIa-6 (6.51 mg, 20%) as a yellowsolid. LC-MS m/z 1461 [M+H]⁺.

Example 16 Dimer IIb-8

This example pertains to FIGS. 16A and 16B and the synthesis of dimerIIb-8.

To a solution of compound 19 (10 g, 21.44 mmol) in acetone (80 mL) wasadded benzyl bromide (5.23 ml, 44.0 mmol, Aldrich) followed by K₂CO₃(11.85 g, 86 mmol, Aldrich). The resulting bright yellow reactionmixture was stirred at 80° C. overnight. The yellow reaction mixture waspoured into 200 mL of cold water. The solid precipitate was collected byfiltration, washed with water and ether, and dried under vaccume to givea light yellow solid (12.8 g, 92%).

To a suspension of the precipitate from the previous step (12.8 g, 19.80mmol) in THF (75 ml) and MeOH (25 mL) was added NaOH (39.6 ml, 119 mmol,3.0 N). The reaction mixture was stirred at RT overnight to give a lightbrown homogeneous solution The reaction mixture was concentrated invacuo to romove most of the organic solvents. The residue wasneutralized with 1.0 N HCl to pH 2-3. The solid formed was collected byfiltration, washed with water and ether to give an off-white solid. Thesolid was dried under vacuum overnight to give compound 92 as a whitesolid (10.21 g, 83%). ¹H NMR (400 MHz, CD3OD) δ 7.47 (s, 2H), 7.42-7.36(m, 10H), 7.16 (s, 2H), 5.18 (s, 4H), 4.31 (t, J=5.9 Hz, 4H), 2.41 (t,J=5.9 Hz, 2H). MS (ESI⁺) m/z 619.5 (M +H)⁺.

Compound 92 was converted to compound 95, proceeding via compounds 93and 94, following the procedures described in Example 14 and FIG. 4A.

To a pressure flask containing Pd(OH)₂ on carbon (0.060 g, 0.085 mmol,Aldrich) was added a solution of compound 95 (0.94 g, 0.853 mmol) inMeOH (10 mL) and EtOAc (10 mL). The resulting reaction mixture wasstirred under H₂ at 20 psi pressure for 2 h and at 40 psi pressure foranother 2 h. The reaction mixture was filtered through a pad of CELITE™,washing with EtOAc. The filtrate was concentrated in vacuo to give 650mg of compound 96 as a faint orange solid (650 mg, 83%). MS (ESI⁺) m/z921.6 (M +H)⁺.

To a solution of compound 96 (101 mg, 0.110 mmol) and 1,4-dibromobutane96a (189 mg, 0.877 mmol, Aldrich) in DMF (1 mL) was added K₂CO₃ (45.5mg, 0.329 mmol). The reaction mixture was stirred at RT overnight. Thereaction was diluted with water. The solid formed was collected byfiltration, and purified by flash chromatography to give compound 97, asa semi solid (110 mg, 84%). MS (ESI⁺) m/z 1191.6 (M+H)⁺.

To a solution of compound 97 (470 mg, 0.395 mmol) and tert-butyl(4-(aminomethyl)phenyl)carbamate 97a (88 mg, 0.395 mmol, Aldrich) in DMF(4 mL) was added K₂CO₃ (164 mg, 1.184 mmol). The reaction was heated at85° C. for 3 h. The reaction was diluted with water and extracted withDCM (3×). The combined organic extracts were dried and concentrated, andpurified by flash chromatography to give compound 98, as a semi solid(175 mg, 35%). MS (ESI⁺) m/z 1251.5 (M +H)⁺.

To a solution of compound 98 (78.5 mg, 0.063 mmol) and 2,6-lutidine(0.022 mL, 0.188 mmol, Aldrich) in DCM (1.0 mL) at RT was addedtrimethylsilyl trifluoromethane sulfonate (TMS-OTf, 0.034 mL, 0.188mmol, Aidrich). The reaction mixture was stirred at RT for 1 h. Thereaction mixture was diluted with DCM and washed with aq. sat. NaHCO₃.The organic phase was dried, concentrated, and purified by flashchromatography to give compound 99, as a semi solid (23 mg, 32%). MS(ESI⁺) m/z 1152.6 (M +H)⁺.

To a −78° C. solution of compound 99 (46 mg, 0.040 mmol) in THF (1 mL)was added a solution of SUPER-HYDRIDE® (0.399 mL, 0.399 mmol, 1M in THF,Aldrich). The reaction was stirred at −78° C. for 1 h. The reaction wasquenched with water and extracted with chloroform (2×), then 10% MeOH inchloroform (2×). The combined organic extracts were dried, concentratedand purified by preparative HPLC. Fractions containing the product wereneutralized with NaHCO₃, and extracted with chloroform (2×) and 10% MeOHin chloroform (2×). The combined organic extracts were dried over MgSO₄,filtered, and concentrated in vacuo. The product was then placed underhigh vacuum over a weekend to give dimer IIb-8 a white solid (12 mg,32%). ¹H NMR (400 MHz, CDCl₃) δ 7.61-7.55 (m, 4H), 7.49 (d, J=7.9 Hz,2H), 7.42-7.31 (m, 6H), 7.09 (d, J=7.9 Hz, 2H), 6.86-6.82 (m 2H), 6.55(s, 2H), 5.03 (d, J=15.5 Hz, 2H), 4.57 (d, J=15.5 Hz, 2H), 4.37-4.29 (m,8H), 4.01-3.88 (m, 4H), 3.37-3.07 (m, 4H), 2.52 (t, J=5.9 Hz, 2H),1.93-1.85 (m 6H), 1.71-1.60 (m, 8H). MS (ESI⁺) m/z 859.2 (M +H)⁺.

Example 17 Additional Dimers and Dimer-Linkers

Following the synthetic principles described hereinabove, the followingadditional dimers and dimer linkers were prepared:

Dimer IIa-2: LCMS (M+H)=615.2 ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.68 (d,J=4.4 Hz, 2H), 7.54 (s, 2H), 6.88 (s, 2H), 4.39-4.25 (m, 4H), 4.23-4.15(m, 2H), 4.14-4.06 (m, 2H), 3.83 (ddd, J=11.7, 7.2, 4.3 Hz, 2H), 3.76(dt, J=7.6, 4.0 Hz, 2H), 3.65-3.55 (m, 2H), 2.40-2.29 (m, 6H), 2.13-2.01(m, 4H), 1.84-1.7 (m, 4H), 1.64-1.52 (m., 4H), 1.48-1.42 (m, 4H).

Dimer IIa-5: LCMS (M+H)=657.4 ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.69 (d,J=4.4 Hz, 2H), 7.55 (s, 2H), 6.84 (s, 2H), 4.36-4.17 (m, 6H), 4.15-4.08(m, 2H), 3.84 (ddd, J=11.7, 7.2, 4.3 Hz, 2H), 3.78-3.72 (m, 2H),3.67-3.56 (m, 2H), 2.46-2.30 (m, 6H), 2.16-2.03 (m, 4H), 1.88-1.78 (m,4H), 1.65-1.50 (m, 6H), 1.48-1.34 (m, 8H).

Dimer IIa-6: LCMS (M+H)=671.4 ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.68 (d,J=4.4 Hz, 2H), 7.53 (s, 2H), 6.84 (s, 2H), 4.34-4.15 (m, 6H), 4.12-4.05(m, 2H), 3.84 (ddd, J=11.7, 7.2, 4.3 Hz, 2H), 3.78-3.73 (m, 2H),3.66-3.56 (m, 2H), 2.52-2.30 (m, 6H), 2.17-2.02 (m, 4H), 1.92-1.77 (m,4H), 1.65-1.50 (m, 6H), 1.43-1.27 (m, 10 H).

Dimer IIa-7: LCMS (M+H)=685.3 ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.68 (d,J=4.4 Hz, 2H), 7.54 (s, 2H), 6.83 (s, 2H), 4.22-4.03 (m, 10H), 3.84(ddd, J=11.7, 7.1, 4.5 Hz, 3H), 3.76 (dt, J=7.5, 4.0 Hz, 2H), 3.61 (dt,J=11.8, 7.8 Hz, 2H), 2.34 (td, J=6.7, 2.6 Hz, 4H), 2.08 (d, J=5.1 Hz,4H), 1.99-1.92 (m, 5H), 1.88-1.76 (m, 8H), 1.59-1.50 (m, 14H), 1.47-1.38(m, 7H).

Dimer IIa-8. LCMS (M+H)=699.5 ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.68 (d,J=4.4 Hz, 2H), 7.52 (s, 2H), 6.81 (s, 2H), 4.23-4.01 (m, 10H), 3.84(ddd, J=11.7, 7.2, 4.3 Hz, 2H), 3.78-3.71 (m, 2H), 3.61 (dt, J=11.9, 7.6Hz, 2H), 2.34 (td, J=6.7, 2.9 Hz, 4H), 2.09 (dd, J=6.9, 4.7 Hz, 4H),2.00-1.92 (m, 5H), 1.88-1.72 (m, 8H), 1.58 (br. s., 13H), 1.39 (d, J=6.2Hz, 12H).

Dimer IIa-10: LC-MS m/z 665 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.68 (d,J=4.4 Hz, 2H), 7.50 (s, 2H), 6.84 (s, 2H), 5.47-5.43 (m, 2H), 5.23-5.14(m, 4H), 4.34-4.20 (m, 8H), 4.18-4.11 (m, 2H), 4.10-4.02 (m, 2H),3.93-3.86 (m, 2H), 3.18-3.08 (m, 2H), 2.99-2.90 (m, 2H), 2.40-2.29 (m,2H), 2.09-2.01 (m, 4H), 1.87-1.76 (m, 4H), 1.64-1.56 (m, 4H).

Dimer IIa-11: LC-MS m/z 667 [M+H]⁺.

Dimer IIa-12: LC-MS m/z 639 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.68 (d,J=4.4 Hz, 2H), 7.51 (s, 2H), 6.86 (s, 2H), 5.22-5.15 (m, J=4.0 Hz, 4H),4.36-4.23 (m, 8H), 4.21-4.14 (m, 2H), 4.12-4.05 (m, 2H), 3.93-3.87 (m,2H), 3.17-3.07 (m, 2H), 2.99-2.90 (m, 2H), 2.34 (quin, J=6.2 Hz, 2H),1.84-1.75 (m, 4H), 1.63-1.54 (m, 4H), 1.48-1.40 (m, 4H).

Dimer IIa-13: LC-MS m/z 643 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d,J=4.4 Hz, 2H), 7.49 (s, 2H), 6.86 (s, 2H), 5.22-5.13 (m, 4H), 4.40-4.25(m, 10H), 4.23-4.17 (m, 2H), 3.94-3.85 (m, 6H), 3.84-3.77 (m, 4H),3.18-3.05 (m, 2H), 2.97-2.89 (m, 2H), 2.32 (quin, J=6.0 Hz, 2H).

Dimer IIa-14: LC-MS m/z 672.5 [M+H2O+H]⁺.

Dimer IIb-1: LC-MS m/z 739 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.55 (s,2H), 7.49 (d, J=5.3 Hz, 2H), 7.41-7.30 (m, 8H), 6.87 (s, 2H), 5.02 (d,J=15.6 Hz, 2H), 4.57 (d, J=15.6 Hz, 2H), 4.38-4.24 (m, 4H), 4.24-4.16(m, 2H), 4.10 (dt, J=9.7, 5.1 Hz, 2H), 4.01-3.94 (m, 2H), 3.34-3.24 (m,2H), 3.22-3.11 (m, 2H), 2.35 (quin, J=6.1 Hz, 2H), 1.91-1.74 (m, 4H),1.59 (d, J=5.9 Hz, 6H), 1.50-1.40 (m, 4H), 0.97-0.82 (m, 2H).

Dimer IIb-2: LC-MS m/z 767 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.53 (s,2H), 7.47 (d, J=5.3 Hz, 2H), 7.39-7.29 (m, 8H), 6.81 (s, 2H), 5.01 (d,J=15.6 Hz, 2H), 4.56 (d, J=15.6 Hz, 2H), 4.33-4.14 (m, 6H), 4.11-4.04(m, 2H), 3.99-3.93 (m, 2H), 3.31-3.24 (m, 2H), 3.19-3.13 (m, 2H), 2.39(quin, J=6.7 Hz, 2H), 1.83-1.75 (m, 4H), 1.62-1.52 (m, 4H), 1.41-1.32(m, 8H).

Dimer IIb-3: LC-MS m/z 739 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.27 (d,J=7.9 Hz, 2H), 7.88 (d, J=4.4 Hz, 2H), 7.56 (s, 2H), 7.32-7.20 (m, 4H),7.14-7.08 (m, 2H), 6.86 (s, 2H), 4.50 (dt, J=10.9, 4.2 Hz, 2H),4.38-4.05 (m, 8H), 3.78-3.67 (m, 2H), 3.55-3.46 (m, 2H), 2.47-2.36 (m,2H), 1.83-1.76 (m, 4H), 1.59 (s, 4H), 1.42-1.31 (m, 8H).

Dimer IIb-4: LC-MS m/z 743.2 (M +H)⁺; ¹H NMR (400 MHz, CDCl₃-d) δ 7.53(s, 2H), 7.47 (d, J=5.1 Hz, 2H), 7.40-7.29 (m, 8H), 6.86 (s, 2H), 5.01(d, J=15.6 Hz, 2H), 4.54 (d, J=15.6 Hz, 2H), 4.37-4.27 (m, 6H),4.24-4.18 (m, 2H), 3.97-3.88 (m, 6H), 3.82 (d, J=1.1 Hz, 4H), 3.31-3.23(m, 2H), 3.19-3.11 (m, 2H), 2.37-2.29 (m, 2H).

Dimer IIb-7. LC-MS m/z 758.6 (M +H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 7.48(s, 2H), 7.43 (d,J=5.3 Hz, 2H), 7.38 (d,J=5.3 Hz, 2H), 7.36-7.26 (m,4H), 7.04 (d, J=8.4 Hz, 1H), 6.87 (s, 2H), 6.52 (br. s., 2H), 4.91 (d,J=15.3 Hz, 2H), 4.53 (d, J=15.0 Hz, 2H), 4.30-4.20 (m, 6H), 4.20-4.16(m, 2H), 3.91-3.88 (m, 6H), 3.65 (s, 4H), 3.46-3.45 (m, 2H), 3.10-3.06(m., 2H), 2.25-2.17 (m, 2H).

Dimer IIc-1: LC-MS m/z 791 [M+H]⁺; ¹H NMR (400 MHz, CHLOROFORM-d) δ 7.91(d, J=3.7 Hz, 2H), 7.56-7.51 (m, 4H), 7.49-7.32 (m, 10H), 6.86 (s, 2H),4.51-4.42 (m, 2H), 4.37-3.84 (m, 8H), 3.68-3.58 (m, 2H), 3.48-3.38 (m,2H), 2.49-2.36 (m, 2H), 1.87-1.74 (m, 4H), 1.69-1.20 (m, 12H).

Dimer IIc-2: LC-MS m/z 907 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.89 (d,J=4.0 Hz, 2H), 7.53 (s, 2H), 7.41-7.39 (m, 2H), 7.37-7.33 (m, 4H),6.94-6.89 (m, 4H), 6.82 (s, 2H), 4.47-4.38 (m, 2H), 4.21-3.96 (m, 8H),3.84 (s, 6H), 3.59 (ddd, J=16.2, 11.4, 1.9 Hz, 2H), 3.39 (ddd, J=16.4,5.2, 1.5 Hz, 2H), 2.03-1.92 (m, 4H), 1.86-1.70 (m, 6H), 1.62-1.51 (m,4H), 1.45-1.30 (m, 12H).

Dimer IIc-3: LC-MS m/z 996 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.89 (d,J=4.0 Hz, 2H), 7.52 (s, 2H), 7.42-7.38 (m, 2H), 7.35-7.32 (m, 4H),6.97-6.91 (m, 4H), 6.82 (s, 2H), 4.46-4.38 (m, 2H), 4.20-4.03 (m, 12H),3.80-3.75 (m, 4H), 3.63-3.54 (m, 2H), 3.48-3.47 (m, 6H), 3.42-3.34 (m,2H), 2.04-1.92 (m, 4H), 1.86-1.71 (m, 6H), 1.60-1.51 (m, 4H), 1.45-1.29(m, 12H).

Dimer IIc-4: LC-MS m/z 1017 [M+H]⁺; ¹H NMR (400 MHz, CHLOROFORM-d) δ7.88 (d, J=4.0 Hz, 2H), 7.52 (s, 2H), 7.39 (s, 2H), 7.33 (d, J=8.6 Hz,4H), 6.91 (d, J=9.0 Hz, 4H), 6.82 (s, 2H), 4.46-4.37 (m, 2H), 4.21-4.01(m, 8H), 3.91-3.85 (m, 8H), 3.63-3.53 (m, 2H), 3.42-3.35 (m, 2H),3.23-3.16 (m, 8H), 1.97 (quin, J=6.9 Hz, 4H), 1.86-1.72 (m, 6H), 1.56(s, 4H), 1.44-1.22 (m, 12H).

Dimer IIc-5: LC-MS m/z 1061 [M+H₂O]⁺; ¹E NMR (400 MHz, CHLOROFORM-d) δ7.88 (d, J=4.0 Hz, 2H), 7.55-7.51 (m, 2H), 7.38 (s, 2H), 7.31 (d, J=8.8Hz, 4H), 6.92 (d, J=8.8 Hz, 4H), 6.81 (s, 2H), 4.45-4.37 (m, 2H),4.20-4.01 (m, 8H), 3.62-3.52 (m, 2H), 3.43-3.33 (m, 2H), 3.30-3.23 (m,8H), 2.63-2.57 (m, 8H), 2.37 (s, 6H), 1.97 (quin, J=7.0 Hz, 4H),1.87-1.71 (m, 6H), 1.66-1.48 (m, 4H), 1.43-1.22 (m, 12H).

Dimer IIc-6: LC-MS m/z 767 [M+H]⁺.

Dimer-linker IIIa-7: LC-MS m/z 777 [M+2H]+.

Dimer-linker IIIa-8: LC-MS m/z 1352.8 [M+H]⁺.

Dimer-linker IIIa-9: LC-MS m/z 1734.2 [M+H]⁺.

Dimer-linker IIIa-10: LC-MS m/z 1633 [M+H]⁺.

Dimer-linker IIIa-12: LC-MS m/z 1707 [M+H]⁺.

Example 18 Biological Activity (Dimers)

The cytotoxic activity of dimers of this invention against variousdifferent cancer cell lines is shown in Table 4. H₂₂₆ is a human lungcancer cell line. N87 is a human gastric cancer cell line. OVCAR3 is ahuman ovarian cancer cell line. HCT116 is a human colon cancer cellline. HCT116/VM46 is a human colon cancer cell line that is multi-drugand paclitaxel resistant.

TABLE 4 Cytotoxic Activity of Dimers Cell Line (IC₅₀, nM) HCT116/ DimerH226 N87 OVCAR3 HCT116 VM46 IIa-1 8.1 4.6 3.4 — 74 IIa-2 1.6 2.7 1.2 — —IIa-3 8.7 8.3 7.3 — — IIa-4 12 22 20 — — IIa-5 69 150 78 — — IIa-6 110310 140 — — IIa-7 12 87 14 — — IIa-8 10 15 9.9 — — IIa-9 12 13 12 — —IIa-10 2.0 2.3 0.47 — 11 IIa-11 13 18 8.6 — 48 IIa-12 0.32 0.89 0.52 —4.8 IIa-13 29 40 20 32 85 IIa-14 21 23 50 38 170 IIb-1 0.22 0.53 0.33 —1.3 IIb-2 3.0 2.5 1.3 — 17 IIb-3 5.9 11 3.5 — 13 IIb-4 0.15 0.14 0.200.068 2.0 IIb-5 0.45 0.35 0.60 0.077 7.3 IIb-6 0.21 0.25 0.21 0.016 0.66IIb-7 0.56 0.40 0.56 0.19 3.5 IIb-8 0.13 0.27 0.25 0.015 1.2 IIc-1 12 127.6 — 30 IIc-2 220 230 >250 48 >250 IIc-3 32 29 104 13 58 IIc-4 — — — —— IIc-5 — — — — — IIc-6 1.1 1.5 1.7 0.13 6.4 IIc-7 0.99 0.62 1.8 0.171.1 IIc-8 0.047 0.034 0.35 0.006 0.076 IIc-9 0.33 0.68 1.0 0.063 0.16IIc-10 0.044 0.36 0.36 0.022 0.071 IIc-11 0.36 0.56 0.31 0.063 0.36IId-1 0.66 1.4 1.2 0.14 1.2 IId-2 0.56 1.1 0.71 0.11 2.8 IId-3 0.0350.072 0.26 0.045 0.033 IId-4 8.1 12 20 2.0 4.8

Example 19 Biological Activity (ADCs)

FIG. 17 shows the acvtivity of two ADCs made with dimer-linker IIIa-7,one with an anti-CD70 antibody and one with an anti-mesothelin antibody.The ADCs were prepared following the procedure generally describedabove. Each had drug-antibody ratio of about 2. Activity was measuredusing a ³H thymidine incorporation assay, where a decrease in theincorporation of the radiolabeled thymidine indicates inhibition of cellproliferation (Cong et. al., U.S. Pat. No. 8,980,824 B2 (2015)). As canbe seen from the figure, both ADCs were active, with EC₅₀ values in therange of nanomolar or less.

Example 20 Comparative Activity

Table 5 compares the cytotoxic activities of dimers of this inventionagainst that of a non-macrocyclic PBD having a structure shown byformula A-4. It is noteworthy that the macrocyclic ring does not appearto introduce conformational constraints that interfere with the abilityof both benzodiazepine rings to slide into the DNA minor groove, asevidence by the comparable, and in some cases, superior cytotoxicpotency.

TABLE 5 Comparative Activities Cell line (EC₅₀, nM) Dimer H226 N87OVCAR3 A-4 70 63 56 IIa-1 8.1 4.6 3.4 IIa-2 1.6 2.7 1.2 IIa-3 8.7 8.37.3 IIa-4 12 22 20 IIa-5 69 150 78 IIa-6 110 310 140 IIa-7 12 87 14IIa-8 10 15 9.9 IIa-9 12 13 12

The foregoing detailed description of the invention includes passagesthat are chiefly or exclusively concerned with particular parts oraspects of the invention. It is to be understood that this is forclarity and convenience, that a particular feature may be relevant inmore than just the passage in which it is disclosed, and that thedisclosure herein includes all the appropriate combinations ofinformation found in the different passages. Similarly, although thevarious figures and descriptions herein relate to specific embodimentsof the invention, it is to be understood that where a specific featureis disclosed in the context of a particular figure or embodiment, suchfeature can also be used, to the extent appropriate, in the context ofanother figure or embodiment, in combination with another feature, or inthe invention in general.

Further, while the present invention has been particularly described interms of certain preferred embodiments, the invention is not limited tosuch preferred embodiments. Rather, the scope of the invention isdefined by the appended claims.

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What is claimed is:
 1. A benzodiazepine dimer having a structurerepresented by formula I

X is

X¹ is CH₂, O, NH, S(O)₀₋₂, 3- to 7-membered cycloalkylene orheterocycloalkylene unsubstituted or substituted with (CH₂)₀₋₅X² orO(CH₂)₂₋₅X², or 5- to 6-membered arylene or heteroarylene unsubstitutedor substituted with (CH₂)₀₋₅X² or O(CH₂)₂₋₅X²; each X² is independentlyMe, CO₂H, NH₂, NH(C₁-C₅ alkyl), N(C₁-C₅ alkyl)₂, SH, CHO,N(CH₂CH₂)₂N(C₁-C₃ alkyl), N(CH₂CH₂)₂NH, NHNH₂, or C(═O)NHNH₂; Y is(CH₂)₄₋₆CH═CH(CH₂)₄₋₆, (CH₂)₄₋₆X¹(CH₂)₄₋₆, or (CH₂)₂(OCH₂CH₂)₂₋₃; eachR¹ and R² is independently H, F, Cl, Br, OH, C₁-C₃ alkyl, O(C₁-C₃alkyl), cyano, (CH₂)₀₋₅NH₂, or NO₂; each double line

in a diazepine ring system independently represents a single bond or adouble bond; each R³ is H if the double line

to the N to which it is attached is a single bond and is absent if thedouble line is a double bond; each R⁴ is H, OH, SO₃Na, or SO₃K if thedouble line

to the C to which it is attached is a single bond and is absent if thedouble line is a double bond; A and B are independently according toformula Ia or Ib

wherein, in formula Ia Y′ and Y″ are independently absent, CH₂, C═O, orCHR¹²; wherein each R¹² is independently F, Cl, Br, or C₁-C₃ alkyl, withthe proviso that Y′ and Y″ are not both absent; each G is independentlyC or N, with the proviso that no more than two Gs are N; and each R⁵,R⁶, R⁷, and R⁸ is independently H, C₁-C₅ alkyl, C≡C(CH₂)₁₋₅X², OH,O(C₁-C₅ alkyl), cyano, NO₂, F, Cl, Br, O(CH₂CH₂O)₁₋₈(C₁₋₃ alkyl),(CH₂)₀₋₅X², O(CH₂)₂₋₅X², 3- to 7-membered cycloalkyl or heterocycloalkylunsubstituted or substituted with (CH₂)₀₋₅X² or O(CH₂)₂₋₅X2, 5- to6-membered aryl or heteroaryl unsubstituted or substituted with(CH₂)₀₋₅X² or O(CH₂)₂₋₅X²,

or where a R⁵, R⁶, R⁷, or R⁸ is attached to a G that is N, such R⁵, R⁶,R⁷, or R⁸ is absent; and wherein, in formula Ib, the dotted linesindicate the optional presence of a C1-C2, C2-C3, or C2-R¹⁰ double bond;R¹⁰ is H, ═O, ═CH₂, ═CH(C₁-C₅ alkyl), CH═CH(CH₂)₁₋₅X², C≡C(CH₂)₁₋₅X²,C₁-C₅ alkyl, OH, O(C₁-C₅ alkyl), cyano, NO₂, F, Cl, Br,O(CH₂CH₂O)₁₋₈(C₁₋₃ alkyl), (CH₂)₀₋₅X², 4- to 7-membered aryl,heteroaryl, cycloalkyl, or heterocycloalkyl unsubstituted or substitutedwith (CH₂)₀₋₅X², O(CH₂)₂₋₅X², 3- to 7-membered cycloalkyl orheterocycloalkyl unsubstituted or substituted with (CH₂)₀₋₅X² orO(CH₂)₂₋₅X², 5- to 6-membered aryl or heteroaryl unsubstituted orsubstituted with (CH₂)₀₋₅X² or O(CH₂)₂₋₅X²; and R⁹ is absent if a C1-C2,C2-C3, or C2-R¹⁰ double bond is present and otherwise is H; or apharmaceutically acceptable salt thereof.
 2. A benzodiazepine dimeraccording to claim 1, having a structure represented by formula IIa:

wherein x is 3 or 5; Y is (CH₂)₇₋₁₂, (CH₂)₂(OCH₂CH₂)₁₋₃,(CH₂)₂₋₄NH(CH₂)₂₋₄ or (CH₂)₂₋₄NH((CH₂)₀₋₁phenyl)(CH₂)₂₋₄ where thephenyl group is optionally substituted with NH₂; A is A1, A2, or A3

and B is B1, B2, or B3


3. A benzodiazepine dimer according to claim 1, having a structurerepresented by formula IIb:

wherein x is 3 or 5; each Y′ is independently absent or CH₂; Y is(CH₂)₇₋₁₂, (CH₂)₂(OCH₂CH₂)₁₋₃, (CH₂)₂₋₄NH(CH₂)₂₋₄ or(CH₂)₂₋₄NH((CH₂)₀₋₁phenyl)(CH₂)₂₋₄ where the phenyl group is optionallysubstituted with NH₂; and R⁴⁰ and R⁴¹ are independently H, Cl, Br, OH,O(C₁₋₃ alkyl), NH₂, or C₁₋₃ alkyl.
 4. A benzodiazepine dimer accordingto claim 1, having a structure represented by formula IIc

wherein x is 3 or 5; Y is (CH₂)₇₋₁₂, (CH₂)₂(OCH₂CH₂)₁₋₃,(CH₂)₂₋₄NH(CH₂)2-4 or (CH₂)₂₋₄NH((CH₂)₀₋₁phenyl)(CH₂)₂₋₄ where thephenyl group is optionally substituted with NH₂; and R⁴² and R⁴³ areindependently H, OMe, NH₂, OCH₂CH₂OMe, N(CH₂CH₂)O, N(CH₂CH₂)NMe, orN(CH₂CH₂)NH.
 5. A benzodiazepine dimer according to claim 1, having astructure represented by formula IIb-6, IIc-8, IIc-9, IIc-10, IIc-11,IId-1, IId-2, or IId-3.
 6. A benzodiazepine dimer-linker compound havinga structure represented by formula III:

wherein R⁶⁰ is according to formula IIIa, IIIa′, or IIIa″

Y is (CH₂)₆₋₁₀; x is 3 or 5; each y is independently 2, 3, or 4; A and Bare independently according to formula Ia or Ib

wherein, in formula Ia Y′ and Y″ are independently absent, CH₂, C═O, orCHR¹²; wherein each R¹² is independently F, Cl, Br, or C₁-C₃ alkyl, withthe proviso that Y′ and Y″ are not both absent; each G is independentlyC or N, with the proviso that no more than two Gs are N; and each R⁵,R⁶, R⁷, and R⁸ is independently H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂,O(C₁₋₃ alkyl), or (OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl); or where a R⁵, R⁶, R⁷, orR⁸ is attached to a G that is N, such R⁵, R⁶, R⁷, or R⁸ is absent; andwherein, in formula Ib, the dotted lines indicate the optional presenceof a C1-C2, C2-C3, or C2-R¹⁰ double bond; R⁹ is absent if a C1-C2,C2-C3, or C2-R¹⁰ double bond is present and otherwise is H; R¹⁰ is H,Cl, Br, ═CH₂, ═CH(C₁₋₅ alkyl), C₁₋₃ alkyl, NO₂, CN, or NH₂; A′ is

R⁵⁰ is H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂, O(C₁₋₃ alkyl), or(OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl) (preferably H); R⁵¹ is H, Cl, Br, C₁₋₃ alkyl,NO₂, CN, or NH₂; T is a self-immolating group; t is 0 or 1; AA^(a) andeach AA^(b) are independently selected from the group consisting ofalanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, asparticacid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,norleucine, norvaline, ornithine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine; p is 1, 2, 3, or 4; q is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10; r is 1, 2, 3, 4, or 5; s is 0 or 1; andR³¹ is


7. A benzodiazepine dimer-linker compound according to claim 6, whereinR⁶⁰ is IIIa.
 8. A benzodiazepine dimer-linker compound according toclaim 6, wherein R⁶⁰ is IIIa′.
 9. A benzodiazepine dimer-linker compoundaccording to claim 6, wherein R⁶⁰ is IIIa″.
 10. A benzodiazepine dimerlinker compound according to claim 6, having a structure represented byformula IIIa-1, IIIa-2, IIIa-3, IIIa-4, IIIa-5. IIIa-6, or IIIa-7.
 11. Aconjugate comprising a benzodiazepine dimer according to claim 1conjugated to an antibody.
 12. A conjugate according to claim 11, havinga structure represented by formula IV:

wherein Ab is an antibody; M is 1, 2, 3, or 4; R⁴⁰ is

where the open valence of R⁴⁰ that is bonded to Ab is denoted by anasterisk (*) and the open valence of R⁴⁰ that is bonded to (CH₂)_(r) isdenoted by a wavy line

; R⁶⁰ is according to formula IIIa, IIIa′, or IIIa″

Y is (CH₂)₆₋₁₀; x is 3 or 5; each y is independently 2, 3, or 4; A and Bare independently according to formula Ia or Ib

wherein, in formula Ia Y′ and Y″ are independently absent, CH₂, C═O, orCHR¹²; wherein each R¹² is independently F, Cl, Br, or C₁-C₃ alkyl, withthe proviso that Y′ and Y″ are not both absent; each G is independentlyC or N, with the proviso that no more than two Gs are N; and each R⁵,R⁶, R⁷, and R⁸ is independently H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂,O(C₁₋₃ alkyl), or (OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl) (preferably H); or where aR⁵, R⁶, R⁷, or R⁸ is attached to a G that is N, such R⁵, R⁶, R⁷, or R⁸is absent; and wherein, in formula Ib, the dotted lines indicate theoptional presence of a C1-C2, C2-C3, or C2-R¹⁰ double bond; R⁹ is absentif a C1-C2, C2-C3, or C2-R¹⁰ double bond is present and otherwise is H;R¹⁰ is H, Cl, Br, ═CH₂, ═CH(C₁₋₅ alkyl), C₁₋₃ alkyl, NO₂, CN, or NH₂; A′is

R⁵⁰ is H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂, O(C₁₋₃ alkyl), or(OCH₂CH₂)₁₋₂O(C₁₋₃ alkyl); R⁵¹ is H, Cl, Br, C₁₋₃ alkyl, NO₂, CN, NH₂; Tis a self-immolating group; t is 0 or 1; AA^(a) and each AA^(b) areindependently selected from the group consisting of alanine, β-alanine,γ-aminobutyric acid, arginine, asparagine, aspartic acid,γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine,norvaline, ornithine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine; p is 1, 2, 3, or 4; q is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10; r is 1, 2, 3, 4, or 5; and s is 0 or
 1. 13. Aconjugate according to claim 12, wherein R⁶⁰ is IIIa.
 14. A conjugateaccording to claim 12, wherein R⁶⁰is IIIa′.
 15. A conjugate according toclaim 12, wherein R⁶⁰ is IIIa″.
 16. A pharmaceutical formulationcomprising a conjugate according to claim 12 and a pharmaceuticallyacceptable excipient.
 17. A method of treating a cancer in a subjectsuffering from such cancer, comprising administering to the subject atherapeutically effective amount of a conjugate of a benzodiazepinedimer according to claim
 12. 18. A method according to claim 17, whereinthe cancer is lung, gastric or ovarian cancer.